Compounds for the treatment of diseases linked to mitochondrial reactive oxygen species (ROS) production

ABSTRACT

The present invention relates to the use of compounds for the prevention and treatment of diseases whose initiation and/or evolution relates to the production and effects of reactive oxygen species (ROS) of mitochondrial origin.

FIELD OF INVENTION

The present invention relates to the use of compounds for the preventionand treatment of diseases whose initiation and/or evolution relate tothe production and effects of reactive oxygen species (ROS) ofmitochondrial origin.

BACKGROUND OF INVENTION

Mitochondrion is at the heart of the now widely acknowledged “freeradical theory of ageing” and thus involved in the pathogenesis ofnearly all ageing-associated diseases, including cardiovascular disease,neurodegenerative diseases (Parkinson's disease, Alzheimer's disease andthe like), cancer and diabetes, as well as tissue dysfunctions ofischemic origin. This theory states that the accumulation of damagescaused by reactive oxygen species (ROS) impacts numerous cellularfunctions, in particular mitochondrial functions, which are essentialfor energy supply and optimal cellular functioning. Mitochondria thusappear as the primary targets of ROS since optimal cellular functioningis crucial for providing the energy for a cell to repair itself.

Interestingly, mitochondria are the major source of reactive oxygenspecies (ROS) and are thus particularly targeted by oxidative damage.Consequently, mitochondrial self-production of ROS causes oxidativedamage that contributes to mitochondrial dysfunction and cell death.

Various antioxidants have been tested with regard to the physiologicaland pathological roles of ROS. Antioxidant research has providednumerous natural and designed molecules that modulate ROS with variousselectivity against the different origins of ROS, being physiological(cellular signalling) or pathological. However, although ROS have beenrelated to numerous diseases, and antioxidants have shown promises inmany preclinical experiments, nearly all clinical trials ofantioxidant-based therapeutics have shown limited efficacy (Orr et al.,2013. Free Radic. Biol. Med. 65:1047-59).

In addition, several recent studies have also demonstrated that too muchreduction of ROS in cells is deleterious and it appears that an adequatebalance of ROS production is necessary for cell functioning (Goodman etal., 2004 Dec. 1. J. Natl. Cancer Inst. 96(23):1743-50; Bjelakovic G etal., 2007 Feb. 28. JAMA. 297(8):842-57). As a consequence, there is agrowing interest in the selective inhibition of ROS production bymitochondria that would not affect cellular signaling by cytosolic ROSproduction.

As mitochondrial oxidative damage contributes to a wide range of humandiseases, antioxidants designed to be accumulated by mitochondria invivo have been developed. The most extensively studied of thesemitochondria-targeting antioxidants is MitoQ, which contains anantioxidant quinone moiety covalently attached to a lipophilictriphenylphosphonium cation. MitoQ has now been used in a range of invivo studies in rats and mice and in two phase II human trials.Conditions of high ROS production are now better characterized. Itappears that ROS may be produced at multiple sites of the respiratorychain in mitochondria (Quinlan C L et al., 2013 May 23. Redox Biol.1:304-12). Maximal superoxide/H₂O₂ production occurs under conditions ofhigh reduction of electron transporters, mainly quinones, and highvalues of mitochondrial membrane potential. Paradoxically, theseconditions are satisfied when mitochondrial oxidative phosphorylation islow (low muscle contraction) or under low oxygen conditions (hypoxia).

The Applicant demonstrates here that AOL (Anethole trithione) does notact as a classical unspecific antioxidant molecule but moreinterestingly as a direct selective inhibitor of the production ofoxygen radicals (ROS) predominantly at site I_(Q) of complex I of themitochondrial respiratory chain, the main mitochondrial site of ROSproduction and the main responsible site for mitochondrial dysfunctions.In addition, the Applicant demonstrates here that AOL does not affectmitochondrial oxidative phosphorylation suggesting the absence of anyadverse side effects and the possibility to treat and/or preventdiseases related to free oxygen-radicals in a long term manner AOL istherefore the first known drug authorized for human use (FDA-marketingauthorization) that prevents mitochondria from producing ROS at siteI_(Q).

SUMMARY

The present invention relates to an inhibitor of production of reactiveoxygen species (ROS) for treating or for use in the treatment of freeoxygen-radicals related diseases.

In one embodiment, said inhibitor is anethole trithione (AOL).

In one embodiment, said inhibitor inhibits mitochondrial production ofROS.

In a preferred embodiment, said inhibitor inhibits mitochondrialproduction of ROS at site IQ of complex I of mitochondria.

In one embodiment, said free oxygen-radicals related diseases areselected from the group comprising: age-related macular degeneration,Parkinson's disease, Alzheimer's disease, ischemic and reperfusioninjury, pulmonary arterial hypertension, scleroderma, atherosclerosis,heart failure, myocardial infarction, arthritis, pulmonary toxicity,cardiopulmonary diseases, inflammatory diseases, cancer, metastasis,cardiac toxicity of anthracyclines, heart failure regardless of origin,ischemia, heart attack, stroke, thrombosis and embolism, asthma,allergic/inflammatory conditions, bronchial asthma, rheumatoidarthritis, Inflammatory Bowel Disease, Huntington's disease, cognitivedisorders, Progeria, progeroid syndromes, epileptic dementia, preseniledementia, post traumatic dementia, senile dementia, vascular dementia,HIV-1-associated dementia, post-stroke dementia, Down's syndrome, motorneuron disease, amyloidosis, amyloid associated with type 11 diabetes,Creutzfelt-Jakob disease, necrotic cell death, Gerstmann-Strausslersyndrome, kuru and animal scrapie, amyloid associated with long-termhemodialysis, senile cardiac amyloid and Familial AmyloidoticPolyneuropathy, cerebropathy, neurospanchnic disorders, memory loss,aluminum intoxication, reducing the level of iron in the cells of livingsubjects, reducing free transition metal ion levels in mammals, patientshaving toxic amounts of metal in the body or in certain bodycompartments, multiple sclerosis, amyotrophic lateral sclerosis,cataract, diabetes, cancer, liver diseases, skin ageing,transplantation, ototoxic secondary effects of aminoglycosides,neoplasms and toxicity of anti-neoplastic or immunosuppressive agentsand chemicals, innate immune responses, and, Friedreich's Ataxia.

In one embodiment, said inhibitor is for preventing or for use in theprevention of metastasis.

Definitions

In the present invention, the following terms have the followingmeanings:

-   -   “Treating” or “treatment” or “alleviation” refers to both        therapeutic treatment and prophylactic or preventative measures;        wherein the object is to prevent or slow down the targeted        pathologic condition or disease. Those in need of treatment        include those already with the disease as well as those prone to        have the disease or those in whom the disease is to be        prevented. A subject or mammal is successfully “treated” for a        disease or affection or condition if, after receiving the        treatment according to the present invention, the subject or        mammal shows observable and/or measurable reduction in or        absence of one or more of the following: reduction ROS        production; and/or relief to some extent, for one or more of the        symptoms associated with the specific disease or condition;        reduced morbidity and mortality, and improvement in quality of        life issues. The above parameters for assessing successful        treatment and improvement in the disease are readily measurable        by routine procedures familiar to a physician.    -   “Therapeutically effective amount” means level or amount of        agent that is aimed at, without causing significant negative or        adverse side effects to the target, (1) delaying or preventing        the onset of a disease, disorder, or condition related to free        oxygen-radicals; (2) slowing down or stopping the progression,        aggravation, or deterioration of one or more symptoms of the        disease, disorder, or condition related to free        oxygen-radicals; (3) bringing about ameliorations of the        symptoms of the disease, disorder, or condition related to free        oxygen-radicals; (4) reducing the severity or incidence of the        disease, disorder, or condition related to free oxygen-radicals;        or (5) curing the disease, disorder, or condition related to        free oxygen-radicals. A therapeutically effective amount may be        administered prior to the onset of the disease, disorder, or        condition related to free oxygen-radicals, for a prophylactic or        preventive action. Alternatively or additionally, the        therapeutically effective amount may be administered after        initiation of the disease, disorder, or condition related to        free oxygen-radicals, for a therapeutic action.    -   “Pharmaceutically acceptable excipient” refers to an excipient        that does not produce an adverse, allergic or other untoward        reaction when administered to an animal, preferably a human. It        includes any and all solvents, dispersion media, coatings,        antibacterial and antifungal agents, isotonic and absorption        delaying agents and the like. For human administration,        preparations should meet sterility, pyrogenicity, general safety        and purity standards as required by regulatory offices, such as,        for example, FDA Office or EMA.    -   “Subject” refers to an animal, including a human. In the sense        of the present invention, a subject may be a patient, i.e. a        person receiving medical attention, undergoing or having        underwent a medical treatment, or monitored for the development        of a disease. In one embodiment, the subject is a male. In        another embodiment, the subject is a female.    -   “About”: preceding a figure means plus or less 10% of the value        of said figure.

DETAILED DESCRIPTION

One object of the present invention is a method for treating freeoxygen-radicals related diseases in a subject in need thereof comprisingthe administration of an effective amount of an inhibitor ofmitochondrial production of reactive oxygen species (ROS).

Another object of the present invention is an inhibitor of production ofreactive oxygen species (ROS) for treating or for use in the treatmentof free oxygen-radicals related diseases, wherein said inhibitorinhibits mitochondrial production of ROS.

In one embodiment, the inhibitor of the invention does not affectphysiological (cytosolic) ROS production. In one embodiment, thephysiological (cytosolic) ROS production is not modulated by more than5% (increase or decrease) in presence of the inhibitor of the invention.

The term “does not affect” as used herein refers to an absence of effectof the inhibitor of the invention measured by technics known to theskilled artisan for determining the level of ROS production.

In another embodiment, the inhibitor of the invention is not aninhibitor of cytosolic ROS production.

Cytosolic ROS production is determined by the difference between totalcellular ROS production and mitochondrial ROS production.

In another embodiment, the inhibitor of the invention acts upstream fromROS production.

Tests to detect cytosolic ROS production are well known in the state ofthe art and to the skilled artisan.

Examples of such tests include:

-   -   Measurement of global cellular ROS production:        5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein        diacetate, acetyl ester (CM-H2DCFDA) and/or H2DCFDA are        indicators for cytosolic reactive oxygen species (ROS) in cells.        CM-H2DCFDA passively diffuses into cells, where its acetate        groups are cleaved by intracellular esterases and its        thiol-reactive chloromethyl group reacts with intracellular        glutathione and other thiols. Subsequent oxidation yields a        fluorescent adduct that is trapped inside the cell, thus        facilitating long-term studies (Zhang, X. et al., 2008. J.        Cardiovasc. Pharmacol. 51(5):443-449; Sarvazyan, N., 1996.        Am. J. Physiol. 271(5 Pt 2):H2079-2085).    -   Measurement of mitochondrial ROS production in cells: Measuring        intracellular ROS in intact cells and assigning the origin to        mitochondria are far more difficult. In recent years, the        proton-motive force crucial to mitochondrial function has been        exploited to target a variety of compounds to the highly        negative mitochondrial matrix using the lipophilic        triphenylphosphonium cation TPP(+) as a “delivery” conjugate.        Among these, MitoSOX Red, also called mito-hydroethidine or        mito-dihydroethidium, is prevalently used for mitochondrial ROS        estimation. The TPP(+) moiety of MitoSOX enables the manifold        accumulation of ROS-sensitive hydroethidine in the mitochondrial        matrix and the oxidation of hydroethidine by superoxide gives        rise to a specific fluorescent oxidation product,        2-hydroxyethidium (Zhao, H. et al., 2005. Proc. Natl. Acad. Sci.        USA. 102(16):5727-5732; Polster, B. M. et al., 2014. Methods        Enzymol. 547:225-250).

In one embodiment, the inhibitor of the invention is a selectiveinhibitor of mitochondrial reactive oxygen species production.

The term “selective inhibitor” as used herein also refers to a compoundcapable of inhibiting ROS production at site IQ of complex I, whilehaving minimal effects on ROS production from the remaining sites and onmitochondrial membrane potential (ΔΨm) and oxidative phosphorylation.For example, on isolated mitochondria, in the presence of rotenone (i.e.when ROS production at site IQ is inhibited) and antimycin A (i.e., whenROS are produced mainly by complex III), the EC50 of the compound on theinhibition of ROS production is about 5, 6, 7, 8, 9, 10, 15, 20 timeshigher than in the absence of rotenone.

In one embodiment, the term “selective inhibitor” as used herein refersto a compound capable of inhibiting mitochondrial ROS production at siteIQ of complex I with an EC₅₀ of about 10 μM. In another embodiment, saidcompound does not significantly inhibit cytosolic ROS production in anin vitro assay of NAD(P)H oxidase ROS production.

In another embodiment, the inhibitor or selective inhibitor of theinvention is a selective inhibitor of ROS production at site I_(Q) ofcomplex I of the mitochondrial respiratory chain.

The term “selective inhibitor” as used herein also refers to a compoundcapable of inhibiting ROS production at site I_(Q) of complex I, whilehaving minimal effects on ROS production from the remaining sites and onmitochondrial membrane potential (ΔΨm) and oxidative phosphorylation.For example, on isolated mitochondria, in the presence of rotenone (i.e.when ROS production at site I_(Q) is inhibited), the EC₅₀ of thecompound on the inhibition of ROS production is about 5, 6, 7, 8, 9, 10,15, 20 times higher than in the presence of antimycin A (i.e., when ROSare produced mainly by complex III).

Inhibition of complex I activity by rotenone and the neurotoxin MPP+ hasbeen linked to parkinsonism in both rodents and humans suggesting a linkbetween dysfunctional complex I, ROS production, and neurodegeneration.Compounds that are capable of inhibiting ROS production from complex Imay therefore be useful in therapy.

Tests to detect specifically ROS produced at site I_(Q) of complex I ofthe mitochondrial respiratory chain on isolated mitochondria fromvarious tissues are well known to the skilled artisan.

High-throughput assays for the identification of inhibitors of ROSproduction at defined sites in isolated mitochondria without alsoaltering energy production are also described. The assays identify sitespecific modulators of ROS production while also revealing less specificeffectors like broad-acting antioxidants and various inhibitors ofmitochondrial bioenergetics. Accordingly, inhibitors that discriminatebetween unwanted electron leak onto oxygen (ROS production) at specificsites within the electron transport chain without altering the normalenergy-coupled electron and proton fluxes across the inner mitochondrialmembrane may be identified. Assays adapt standard fluorescence basedassays of mitochondrial ROS production using the dye Amplex UltraRed(Invitrogen) and ΔΨm using the potentiometric dye TMRM (Invitrogen) to ahigh throughput microplate format. A core set of five ROS and one ΔΨmassays for robust detection of functional modulation in freshly isolatedskeletal muscle mitochondria are provided. Five major sites of ROSproduction (site I_(Q), I_(F), III_(QO), SDH, and mGPDH) may be targetedseparately by varying the substrates and inhibitors added to a commonassay mixture. A counterscreen to monitor ΔΨm may be run in parallel toeliminate compounds that are likely general inhibitors or uncouplers ofnormal mitochondrial energy production.

In another embodiment, inhibitors are tested at 2.5 μM in duplicateagainst all assays. Endpoint fluorescence is normalized to DMSO andknown mitochondrial inhibitor control wells included on each plate.Positive hits in each ROS assay may be initially filtered by applying athreshold of preferably 15% or more preferably 18% or even morepreferably 20% or more reduction in that assay. Each ROS assay may beemployed as a counterscreen against the others while also eliminatingcompounds that altered ΔΨm in the TMRM-based counterscreen. Therefore,filtered hits may be subsequently assessed to eliminate those thataltered the other ROS assays by more than e.g., 20% or 18% or 15% or ΔΨmby more than, preferably 10% or more preferably 5% or even morepreferably 4%.

In another embodiment, inhibitors that are selective inhibitors of ROSproduction from a single site of ROS production decrease ROS productionfrom one of the ROS production site I_(Q), I_(F), III_(QO), SDH, andmGPDH by greater than 18%, while affecting ROS production from theremaining sites of ROS production by less than 10%.

Complex I of the respiratory chain can generate ROS from two distinctsites: the ubiquinone binding site and the flavin mononucleotide site.

Ubiquinone Binding Site of Complex I (I_(Q)):

To specifically analyze ROS production from I_(Q), 5 mM succinate may beused as the substrate to supply electrons to the respiratory chain.I_(Q) ROS production is exceptionally sensitive to changes in the protonmotive force (PMF) across the mitochondrial inner membrane(PMF=ΔΨm+ΔpH). Therefore, a conservative threshold for the ΔΨm assaywhen evaluating selectivity of hits in the I_(Q) ROS assay may beutilized.

Electron leak from site I_(Q) is best characterized during reversetransport from a reduced Q-pool to matrix NAD⁺ via CI in the presence ofa strong PMF. Experimentally, conditions that favor I_(Q) ROS productionare considered far removed from physiology leading many to dismiss itsrelevance despite its capacity for high rates. However, even whenprovided with lower concentrations of both glutamate (to feed electronsforward through CI) and succinate (to feed electrons in reverse),respiring mitochondria still produce significant levels ofrotenone-sensitive ROS (i.e. I_(Q) ROS). Further, comparative analysesshow an inverse relationship between maximal ROS production from siteI_(Q) (but not site I_(F)) and maximum life span across diversevertebrate species (Lambert, A. et al., 2007. Aging Cell. 6(5):607-18;Lambert, A. et al., 2010. Aging Cell. 9(1):78-91.). Therefore, selectivemodulators of I_(Q) ROS would offer unique opportunities to probe theputative role of mitochondrial ROS production in normal and pathologicalprocesses.

Flavin Binding Site of Complex I (I_(F)):

To specifically analyze ROS production from I_(F), the substratesolution to supply electrons to the respiratory chain may comprise 5 mMglutamate, 5 mM malate and 4 μM rotenone. Site I_(F) produces ROS at arate proportional to the reduction state of the NADH pool in themitochondrial matrix (Treberg, J. et al., 2011. J. Biol. Chem.286(36):31361-72). Blockade of site I_(Q) with the pesticide rotenonecan increase ROS production from site I_(F) by preventing oxidation ofthe flavin. Maximal ROS production from the flavin binding site ofcomplex I (site I_(F)) is relatively low compared to sites I_(Q) andIII_(QO) and this may lead to higher variability in this assay andsubsequently a higher false positive rate of hit calling in the originalscreen.

In another embodiment, the inhibitor of the invention does not affectsignificantly oxidative phosphorylation directly on mitochondria,preferably oxidative phosphorylation is modulated by less than 10, 9, 8,7, 6, 5%.

Diseases related to free oxygen-radicals relate to oxidative stressimbalances and mitochondrial dysfunction. In particular, diseasesrelated to mitochondrial dysfunctions are induced by mitochondrial ROSproduction.

Diseases related to free oxygen-radicals include but are not limited to:aging diseases, auto-immune diseases, cardiovascular diseases, progeroidsyndromes, Parkinsonian syndromes, neurological diseases, ischemic andreperfusion injuries, infectious diseases, muscles diseases, lung,kidney and liver diseases.

Aging diseases include but are not limited to: age-related maculardegeneration (AMD), skin ageing, UV damage to the skin, thinning,sagging, wrinkling, the appearance of age spots, broken blood vesselsand areas of dryness, seborrhoeic keratosis, solar keratoses, KindlerSyndrome, Bowen's disease, skin cancer, arthritis, ankylosingspondylitis, inflammatory polyarthropathies, knee arthritis, epidemicpolyarthritis, psoriatic arthritis, cataract, deafness, cancer,metastasis, metastasis processes prevention, liver diseases,transplantation, neoplasms and toxicity of anti-neoplastic orimmunosuppressive agents and chemicals, osteoporosis, poikiloderma,acrogeria, hereditary sclerosing poikiloderma, dyskeratosis congenita,xeroderma pigmentosum, Bloom's syndrome, Fanconi anemia, Cockaynesyndrome, and pollution-induced diseases.

Autoimmune diseases include but are not limited to: multiple sclerosis,rheumatoid arthritis, systemic lupus erythematosis, type I diabetesmellitus, Crohn's disease; myasthenia gravis, Grave's disease,scleroderma, Sjogren's syndrome, ulcerative colitis, primary biliarycirrhosis, autoimmune hepatitis, Hashimoto's thyroiditis, ankylosingspondylitis, psoriasis. The autoimmune disease can be an autoimmunedisease related to blood disorders such as autoimmune hemolytic anemia,pernicious anemia and autoimmune thrombocytopenia. The autoimmunedisease can also be temporal areritis, anti-phospholipid syndrome,vasculitides such as Wegener's granulomatosis and Behcet's disease.Other autoimmune diseases include polymyositis, drmatomyositis,spondyloarthropthies such as ankylosing spondylitis, anti-phospholipidsyndrome, and polymyocysitis.

Cardiovascular diseases include but are not limited to: hypertension,cardiac toxicity of anti-cancer drugs, cardiac toxicity ofanthracyclines, cardiac toxicity of quinolones, heart failure regardlessof origin, ischemia, heart attack, stroke, atherosclerosis, cardiacfibrillation, hypertension, thrombosis and embolism,allergic/inflammatory conditions such as bronchial asthma, rheumatoidarthritis, inflammatory Bowel disease, type II diabetes, diabetesmellitus and deafness (DAD) or Ballinger-Wallace syndrome, inflammatorydiseases, rheumatic fever, pulmonary arterial hypertension, innateimmune responses, cardiopulmonary diseases such as: chronic obstructivepulmonary disease, pulmonary embolism, pericarditis, coarctation ofaorta, tetralogy of Fallot, aortic stenosis, mitral stenosis, aorticregurgitation, mitral regurgitation, pneumoconiosis, bronchiectasis,cardiomyopathies, endothelial nitroglycerin tolerance.

Progeroid syndromes include but are not limited to: progeria, Bloomsyndrome, Cockayne syndrome, De Barsy syndrome, dyskeratosis congenita,restrictive dermopathy, Rothmund-Thomson syndrome, trichothiodystrophy,Werner syndrome, Wiedemann-Rautenstrauch syndrome, xerodermapigmentosum.

Parkinsonian syndromes include but are not limited to: Parkinson'sdisease (PD), progressive supranuclear palsy, multiple system atrophy,corticobasal degeneration or Lewy body dementia, toxin-inducedParkinsonism, and an early-onset variant of PD such as an autosomalrecessive PARK6-linked Parkinsonism or an autosomal recessivePINK1-linked Parkinsonism.

Neurologic diseases include but are not limited to: dementia,Alzheimer's disease, Parkinson's disease and ageing, Huntington'sdisease, Friedreich's Ataxia, Wilson's disease, Leigh syndrome,Kearns-Sayre syndrome, Leber hereditary optic neuropathy, cognitivedisorders, mood disorders, movement disorders, tardive dyskinesia, braininjury, apoptosis, dementia, epilepsy, epileptic dementia, preseniledementia, post traumatic dementia, senile dementia, vascular dementia,HIV-1-associated dementia, post-stroke dementia, schizophrenia, Down'ssyndrome, motor neuron disease, amyloidosis, amyloid associated withtype II diabetes, Creutzfelt-Jakob disease, necrotic cell death,Gerstmann-Straussler syndrome, kuru and animal scrapie, amyloidassociated with long-term hemodialysis, senile cardiac amyloid andfamilial amyloidotic polyneuropathy, cerebropathy, neurospanchnicdisorders, memory loss, aluminum intoxication, reducing the level ofiron in the cells of living subjects, reducing free transition metal ionlevels in mammals, patients having toxic amounts of metal in the body orin certain body compartments, multiple sclerosis, amyotrophic lateralsclerosis, akinetopsia, alcohol-related dementia, primary age-relatedtauopathy, anomic aphasia, anosognosia, apraxia, apraxia of speech,auditory verbal agnosia, frontotemporal dementia, frontotemporal lobardegeneration, logopenic progressive aphasia, neurofibrillary tangle,phonagnosia, Pick's disease, primary progressive aphasia, progressivenonfluent aphasia, semantic dementia, steroid dementia syndrome,visuospatial dysgnosia, ototoxic secondary effects of aminoglycosides,cocaine toxicity.

Ischemic and reperfusion injury include but are not limited to: stroke,brain ischemia, brainstem stroke syndrome, carotid endarterectomy,cerebellar stroke syndrome, cerebral achromatopsia, cerebral hemorrhage,cerebral infarction, cerebral venous sinus thrombosis, intraparenchymalhemorrhage, intracranial hemorrhage, lacunar stroke, lateral medullarysyndrome, lateral pontine syndrome, partial anterior circulationinfarct, posterior circulation infarct, silent stroke, trokeAssociation, stroke belt, stroke recovery, transient ischemic attack,Watershed stroke, Weber's syndrome, obesity, organ preservation fortransplantation, ischemia, reperfusion injury.

Infectious diseases include but are not limited to: hepatitis C, sepsis,infectious myopathies, septic shock.

Muscles diseases include but are not limited to: myopathies,mitochondrial myopathies, facioscapulohumeral muscular dystrophy,facioscapulohumeral muscular dystrophy type 1, facioscapulohumeralmuscular dystrophy type 2, Ryanodine Receptor 1 (RYR1) related myopathy,selenoprotein 1 (SEPN1)-related myopathy Kearns-Sayre syndrome,cardiomyopathies, movement disorder, immobilization-induced muscleatrophy, skeletal muscle burn injury, Dupuytren's contracture.

Lung, kidney and liver diseases include but are not limited to: cysticfibrosis, asthma, pollution-induced diseases, cardio-pulmonary diseases,pulmonary arterial hypertension, chronic obstructive pulmonary disease,pulmonary embolism, pneumoconiosis, bronchiectasis, bronchial asthma,ventilator-induced diaphragm dysfunction, lung cancer, alcohol fattyliver disease, fatty liver disease, diabetes, kidney preservation exvivo, liver inflammation in hepatitis C, kidney damage in type Idiabetes, cirrhosis.

Diseases to be treated in particular in the present invention are agingdisease, AMD, skin aging, cardiovascular diseases such as for examplecardiac toxicity of anthracyclines, progeria and progeroid syndromes,Parkinson's disease, Alzheimer's disease, Friedreich's Ataxia, ischemiareperfusion, cardio-pulmonary diseases, asthma, cancer, metastasis,pollution-induced diseases.

In one embodiment, a disease to be particularly prevented in the presentinvention is metastasis. Indeed, ROS production is involved inmechanisms of tumor growth and metastasis: tumor cell migration,invasion, clonogenicity, metastatic take, and spontaneous metastasis arepromoted by the natural selection of a mitochondrial phenotypeassociated with ROS production and aberrant TCA cycle activity, amechanism named “metastatic mitochondrial switch” (Porporato et al.,2014. Cell Reports. 8:754-766). ROS hyper production also promotesangiogenesis and reciprocally inhibitors of ROS production areantiangiogenic products.

In one embodiment, the inhibitor or selective inhibitor of the inventionis of one of the following formulas:

and oxides, derivatives and metabolites thereof, whereinZ is S, O, NR, R₂ or CR₂;R is —H, —OH, C₁-C₅ alkyl, C₁-C₅ alkoxy or C₁-C₅ alkoxycarbonyl;R2, together with the atoms to which it is bonded, comprises a spiroring;R1, R2, R3, and R4 independently are —H, -alkyl, -aryl, -alkylaryl, aheterocycle, a halogen, -alkoxycarbonyl (C₁-C₅) or -carboxyl;wherein either alkyl is a C₁-C₁₀ linear or branched chain, saturated orunsaturated moiety, which is optionally substituted by 1, 2 or moreindependently selected ether (—O—), halogen, alkyl (C₁-C₅), —OH, alkoxy(C₁-C₅), alkoxycarbonyl, (C₁-C₅), carboxyl, amido, alkyl amido (C₁-C₅),amino, mono- or dialkylamino (C₁-C₅), alkyl carbamoyl (C1-C5), thiol,alkylthio (C₁-C₅), or benzenoid aryl; andwherein the -aryl and -alkylaryl substituent for R1, R2, R3 and R4comprises a benzenoid group (C₆-C₁₄), wherein the benzenoid group isoptionally substituted with 1, 2 or more independently selected —SO₃H,halogen, alkyl (C₁-C₅), —OH, alkoxy (C₁-C₅), alkoxycarbonyl, (C₁-C₅),carboxyl, amido, alkyl amido (C₁-C₅), amino, mono- or dialkylamino(C₁-C₅), alkyl carbamoyl (C₁-C₅), thiol, alkylthio (C₁-C₅); andwherein the heterocycle is defined as any 4, 5 or 6 membered, optionallysubstituted heterocyclic ring, saturated or unsaturated, containing 1-3ring atoms selected from N, O and S, the remaining ring atoms beingcarbon; and wherein said substituents on said aryl or said heterocyclicare selected from the group consisting of halogen, alkyl (C₁-C₅),hydroxyl, alkoxy (C₁-C₅), alkoxycarbonyl (C₁-C₅), carboxyl, amido, alkylamido (C₁-C₅), amino, mono and dialkyl amino (C₁-C₅), alkyl carbamoyl(C₁-C₅), thiol, alkylthio (C₁-C₅), benzenoid, aryl, cyano, nitro,haloalkyl (C₁-C₅), alklsulfonyl (C₁-C₅), or sulfonate, orone of R1 and R2 and one of R3 and R4 together with the carbon atoms towhich they are attached comprise a fused bicyclic or tricyclic compound,which is saturated or unsaturated, heterocyclic or carbocyclic andwherein the rings are all optionally substituted 5-, 6-, 7- or8-membered rings, with substituents optionally selected from alkyl,alkoxy, —SO₃H, —OH and halogen, orR1 and R2 together or R3 and R4 together independently are oxime (═NOH).

Examples of 1,2 dithiolane class inhibitors include but are not limitedto: lipoamide (1,2 dithiolane); 1,2-Dithiolane-4-carboxylic acid;4-octyl-1,3-dithiolane-2-thione; 4-decyl-1,3-dithiolane-2-thione;4-dodecyl-1,3-dithiolane-2-thione; 4-tetradecyl-1,3-dithiolane-2-thione;and a 1,3-dithiolane-2-thione.

Examples of 1,2 dithiole class inhibitors include but are not limitedto: 4-methyl-5-(2-pyrazinyl)-3-dithiolethione (oltipraz);5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione (anetholetrithione or AOL),anethole dithiolethione (ADT), ADO, 1,2-dithiole-3-thione;5-(4-phenyl-1,3-butadienyl)-1,2-dithiol-3-thione, 5-4(4-chlorophenyl)-1,3-butadienyl-1,2-dithiol-3-thione,5-{4-(4-methoxyphenyl)-1,3-butadienyl}-1,2-dithiol-3-thione,5-{4-(p-toluyl)-1,3-butadienyl}-1,2-dithiol-3-thione,5-{4-(o-chlorophenyl)-1,3-butadienyl}-1,2-dithiol-3-thione and5-{4-(m-methyl phenyl)-1,3-butadienyl}-1,2-dithiol-3-thione.

Examples of 1,3 dithiole class inhibitors include but are not limitedto: diisopropyl 1,3-dithiol-2-ylidenemalonate (malotilate);1,3-dithiolo(4.5-d)-1,3-dithiino-2-thione;1,3-dithiolo(4.5-d)-1,3-dithiole-2-thione; 5-chloro-1,3-dithiolo(4.5-d)-1,3-dithiole-2-thione; and5-cyano-1,3-dithiolo(4.5-d)-1,3-dithiole-2-thione.

Examples of 1,3 dithiolane class inhibitors include but are not limitedto:5-(1-carbonyl-L-amino-acid)-2,2-dimethyl-[1,3]dithiolane-4-carboxylicacid; Hexahydro-1-3-benzodithiole-2-thione;4-Octyl-1,3-dithiolane-2-thione; 4-Decyl-1,3-dithiolane-2-thione;4-Dodecyl-1,3-dithiolane-2-thione.

The inhibitor or selective inhibitor of the invention is preferablyselected from the group comprising:5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione (anetholetrithione or AOL),anethole dithiolethione (ADT), ADO, 1,2-dithiole-3-thione,1,2-dithiolane, 1,3-dithiole-2-thione,4-methyl-5-(2-pyrazinyl)-3-dithiolethione (oltipraz), and diisopropyl1,3-dithiol-2-ylidenemalonate (malotilate) or derivatives or analogthereof.

Examples of inhibitors or selective inhibitors of the invention includebut are not limited to:

In one embodiment, the inhibitor of the invention is:

5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione (AOL).

In one embodiment, the inhibitor of the invention is not a chelatingagent, preferably, a chelating agent of Fe and/or Cu.

In one embodiment, the inhibitor of the invention is not oltipraz.

In one embodiment, the inhibitors or selective inhibitors are selectedfrom those described in the following Patent Application: US2004/053989.

In another embodiment, the inhibitors or selective inhibitors areselected from those described in the following Patents: U.S. Pat. No.3,040,057; EP0576619; U.S. Pat. Nos. 3,576,821; 3,959,313; 3,109,772.

In one embodiment, the inhibitor is notN-cyclohexyl-4-(4-nitrophenoxy)benzenesulfonamide.

The present invention also relates to a composition for treatingdiseases related to free oxygen-radicals in a subject in need thereof,comprising or consisting of or consisting essentially of the inhibitoras hereinabove described.

The present invention also relates to a composition for treating or foruse in treating free oxygen-radicals related diseases, wherein saidcomposition comprises or consists of or consists essentially of aninhibitor or selective inhibitor of mitochondrial production of ROS.

The present invention also relates to a pharmaceutical composition fortreating diseases related to free oxygen-radicals in a subject in needthereof, comprising or consisting of or consisting essentially of theinhibitor as hereinabove described in combination with at least onepharmaceutically acceptable excipient.

The present invention also relates to a pharmaceutical composition fortreating or for use in treating free oxygen-radicals related diseases,wherein said pharmaceutical composition comprises or consists of orconsists essentially of an inhibitor or selective inhibitor ofmitochondrial production of ROS and at least one pharmaceuticallyacceptable excipient.

The present invention also relates to a medicament for treating diseasesrelated to free oxygen-radicals in a subject in need thereof, comprisingor consisting of or consisting essentially of the inhibitor ashereinabove described.

The present invention also relates to a medicament for treating or foruse in treating free oxygen-radicals related diseases, wherein saidmedicament comprises or consists of or consists essentially of aninhibitor or selective inhibitor of mitochondrial production of ROS.

Suitable excipients include water, saline, Ringer's solution, dextrosesolution, and solutions of ethanol, glucose, sucrose, dextran, mannose,mannitol, sorbitol, polyethylene glycol (PEG), phosphate, acetate,gelatin, collagen, Carbopol®, vegetable oils, and the like. One mayadditionally include suitable preservatives, stabilizers, antioxidants,antimicrobials, and buffering agents, such as, for example, BHA, BHT,citric acid, ascorbic acid, tetracycline, and the like.

Other examples of pharmaceutically acceptable excipients that may beused in the composition of the invention include, but are not limitedto, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

In addition some excipients may include, surfactants (e.g.hydroxypropylcellulose); suitable carriers, such as, for example,solvents and dispersion media containing, for example, water, ethanol,polyol (e.g. glycerol, propylene glycol, and liquid polyethylene glycol,and the like), suitable mixtures thereof, and vegetable oils, such as,for example, peanut oil and sesame oil; isotonic agents, such as, forexample, sugars or sodium chloride; coating agents, such as, forexample, lecithin; agents delaying absorption, such as, for example,aluminum monostearate and gelatin; preservatives, such as, for example,benzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosaland the like; buffers, such as, for example, boric acid, sodium andpotassium bicarbonate, sodium and potassium borates, sodium andpotassium carbonate, sodium acetate, sodium biphosphate and the like;tonicity agents, such as, for example, dextran 40, dextran 70, dextrose,glycerin, potassium chloride, propylene glycol, sodium chloride;antioxidants and stabilizers, such as, for example, sodium bisulfite,sodium metabisulfite, sodium thiosulfite, thiourea and the like;nonionic wetting or clarifying agents, such as, for example, polysorbate80, polysorbate 20, poloxamer 282 and tyloxapol; viscosity modifyingagents, such as, for example dextran 40, dextran 70, gelatin, glycerin,hydroxyethylcellulose, hydroxymethylpropylcellulose, lanolin,methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol,polyvinylpyrrolidone, carboxymethylcellulose; and the like.

In one embodiment, the composition, the pharmaceutical composition orthe medicament of the invention is to be administered systemically orlocally.

In one embodiment, the composition, the pharmaceutical composition orthe medicament of the invention is to be administered orally, byinjection, topically, nasally, buccally, rectally, vaginaly,intratracheally, by endoscopy, transmucosally, and by percutaneousadministration.

In one embodiment, the composition, the pharmaceutical composition orthe medicament of the invention is injected, preferably systemicallyinjected. Examples of formulations adapted to systemic injectionsinclude, but are not limited to: liquid solutions or suspensions, solidforms suitable for solution in, or suspension in, liquid prior toinjection. Examples of systemic injections include, but are not limitedto, intravenous, subcutaneous, intramuscular, intradermal andintraperitoneal injection, and perfusion. In another embodiment, wheninjected, the composition, the pharmaceutical composition or themedicament of the invention is sterile. Methods for obtaining a sterilepharmaceutical composition include, but are not limited to, GMPsynthesis (GMP stands for “Good manufacturing practice”).

In another embodiment, the composition, the pharmaceutical compositionor the medicament of the invention is orally administered. Examples offormulations adapted to oral administration include, but are not limitedto: solid forms, liquid forms and gels. Examples of solid forms adaptedto oral administration include, but are not limited to, pill, tablet,capsule, soft gelatine capsule, hard gelatine capsule, caplet,compressed tablet, cachet, wafer, sugar-coated pill, sugar coatedtablet, or dispersing/or disintegrating tablet, powder, solid formssuitable for solution in, or suspension in, liquid prior to oraladministration and effervescent tablet. Examples of liquid form adaptedto oral administration include, but are not limited to, solutions,suspensions, drinkable solutions, elixirs, sealed phial, potion, drench,syrup and liquor.

In another embodiment, the composition, the pharmaceutical compositionor the medicament of the invention is topically administered. Examplesof formulations adapted to topical administration include, but are notlimited to, sticks, lipsticks, waxes, creams, lotions, ointments, balms,gels, glosses, sunscreen preparations, cosmetics, masks, leave-on washesor cleansers, depilatory preparations and/or the like.

Topical administration characterizes the delivery, administration orapplication of the composition, the pharmaceutical composition or themedicament of the invention directly to the site of interest for alocalized effect (generally onto one or more exposed or outer surfacesthereof, such as the outermost layer of the epidermis, which is exposedand visually observable), for example, using hands, fingers or a widevariety of applicators (roll-up, roll-on or other stick container, tubecontainer, cotton ball, powder puff, Q-tip, pump, brush, mat, clothand/or the like). The application may be made, for example, by laying,placing, rubbing, sweeping, pouring, spreading and/or massaging into, oronto, the skin, or by any other convenient or suitable method.Preferably, topical administration is effected without any significantabsorption of components of the composition into the subject's bloodstream (to avoid a systemic effect).

The composition, the pharmaceutical composition or the medicament of theinvention of the invention can be mixed to form white, smooth,homogeneous, opaque cream or lotion with, for example, benzyl alcohol 1%or 2% (wt/wt) as a preservative, emulsifying wax, glycerin, isopropylpalmitate, lactic acid, purified water and sorbitol solution. Inaddition, the compositions can contain polyethylene glycol 400. They canbe mixed to form ointments with, for example, benzyl alcohol 2% (wt/wt)as preservative, white petrolatum, emulsifying wax, and tenox II(butylated hydroxyanisole, propyl gallate, citric acid, propyleneglycol). Woven pads or rolls of bandaging material, e.g. gauze, can beimpregnated with the compositions in solution, lotion, cream, ointmentor other such form can also be used for topical application.

One object of the present invention is a cosmetic composition comprisingthe inhibitor of the invention.

Another object of the invention is a cosmeceutical compositioncomprising the inhibitor of the invention.

In another embodiment, the composition of the invention can also beapplied topically using a transdermal system, such as one of anacrylic-based polymer adhesive with a resinous crosslinking agentimpregnated with the composition and laminated to an impermeablebacking.

In one embodiment, the composition of the present invention can beadministered as a transdermal patch, more particularly as asustained-release transdermal patch. The transdermal patches can includeany conventional form such as, for example, adhesive matrix, polymericmatrix, reservoir patch, matrix or monolithic-type laminated structure,and are generally comprised of one or more backing layers, adhesives,penetration enhancers, an optional rate controlling membrane and arelease liner which is removed to expose the adhesives prior toapplication. Polymeric matrix patches also comprise a polymeric-matrixforming material. Suitable transdermal patches are described in moredetail in, for example, U.S. Pat. Nos. 5,262,165; 5,948,433; 6,010,715and 6,071,531, the disclosure of each of which are incorporated hereinin their entirety.

Examples of formulations adapted to transdermal administration include,but are not limited to, ointment, paste, cream, film, balm, patch, suchas, for example, transdermal patch, gel, liposomal forms and the like.

In one embodiment, the transdermal composition is an ointment, paste,cream; film, balm, patch, such as, for example, transdermal patch, gel,liposomal forms or the like.

In one embodiment of the invention, the ointment is an oleaginousointment; an emulsified ointment such as, for example, oil-in-water or awater-in-oil ointment; or a water-soluble ointment, preferably is anoleaginous ointment.

In one embodiment of the invention, the oleaginous ointment uses basessuch as, for example, plant and animal oils; plant and animal fats;waxes; vaseline, such as, for example, white vaseline or vaseline oil;and paraffin such as, for example, liquid paraffin or paraffin oil.

In one embodiment of the invention, the transdermal composition furthercomprises one or more excipients. Suitable pharmaceutically acceptableexcipients are well known from the skilled person. Examples of suitableexcipients include, but are not limited to, carriers, emulsifyingagents, stiffening agents, rheology modifiers or thickeners,surfactants, emollients, preservatives, humectants, buffering agents,solvents, moisturizing agents and stabilizers.

In another embodiment, a particular administration route may beintraocularly. In another embodiment, the administration route may be atopical ocular administration, such as, for example, the administrationof eye drops or by bathing the eye in an ophthalmic solution comprisingthe inhibitor of the invention.

The ophthalmic solution refers to sterile liquid, semi-solid or solidpreparations intended for administration upon the eyeball and/or to theconjunctiva, or for insertion in the conjunctival sac or foradministration into the posterior segment of the eye. As used herein,the term “posterior segment of the eye” refers to the back two third ofthe eye, comprising the anterior hyaloids membrane and the structuresbehind it (vitreous humor, retina, choroid, optic nerve). In particular,an ophthalmic composition may be administered into the vitreous, forexample by intravitreous injection. Examples of ophthalmic compositionsinclude, but are not limited to, eye drops, eye lotions, powders for eyedrops and powders for eye lotions, and compositions to be injected intothe conjunctival sac or into the vitreous.

Examples of carriers include, but are not limited to, water; bufferedsaline; petroleum jelly (Vaseline, also known as white soft paraffin);petrolatum; oils, such as, for example, mineral oil, vegetable oil,animal oil, paraffin oil, castor oil or vaseline oil; organic andinorganic waxes, such as, for example, microcrystalline, paraffin, beeswax and ozocerite wax; natural polymers, such as, for example,xanthanes, gelatin, cellulose, collagen, starch, or gum arabic;synthetic polymers; alcohols; polyols; and the like. In one embodimentof the invention, the carrier is a base cream, comprising an emulsifyingagent, an oil-phase ingredient and a water phase ingredient.

Examples of well-known ointment or lotion base excipients include, butare not limited to, Vaseline, Plastibase™ (which is a base prepared withpolyethylene (average molecular weight of about 21000 Da) and liquidparaffin) or ESMA-P™ (made of microcrystalline wax).

Examples of emulsifying agents include, but are not limited to, cetylalcohol; cetostearyl alcohol; stearyl alcohol; carboxypolymethylene;polycarbophil; polyethylene glycol and derivatives thereof;polyoxyethylene and derivatives thereof, such as, for example,polysorbate 20 or polysorbate 80, alone or in combination with fattyalcohols such as, for example, cetyl alcohol, stearyl alcohol andcetostearyl alcohol; and sorbitan esters, such as, for example, sorbitanfatty acid ester.

Examples of oil-phase ingredient include, but are not limited to,Vaseline, such as, for example, white Vaseline, yellow Vaseline orVaseline oil; paraffin such as, for example, liquid paraffin or paraffinoil; dimethicone and mixtures thereof.

Examples of water-phase ingredients include, but are not limited to,water, glycerol and propyleneglycol.

Examples of stiffening agents include, but are not limited to, stearylalcohol, cetostearyl alcohol, and cetyl alcohol.

Examples of rheology modifiers or thickeners include, but are notlimited to, carbomers such as, for example, Carbopol®, andpolyoxyethylene tallow amines such as, for example, Ethomeen®.

Examples of surfactants include, but are not limited to, anionic,cationic, amphoteric, and nonionic surfactants, such as, for example,sodium lauryl sulfate, cetostearyl alcohol, cetyl alcohol, magnesiumlauryl sulfate, a wax, or a combination thereof.

Examples of emollients include, but are not limited to, white or yellowpetrolatum (white or yellow vaseline), liquid petrolatum (liquidvaseline), paraffin, or aquaphor.

Examples of preservatives include, but are not limited to, antimicrobialpreservatives such as, for example, nipagin (methyl hydroxybenzoate),nipasol (hydroxybenzoate), butylparaben, ethylparaben, methylparaben,propyl paraben potassium, propyl paraben sodium; parahydroxybenzoateesters; sorbic acid; potassium sorbate; benzoic acid; parabens;chlorobutanol; phenol; thimerosal; sodium benzoate and benzyl alcohol.

Examples of humectants include, but are not limited to, propylene glycoland propylene glycol alginate.

Examples of buffering agents include, but are not limited to, sodiumhydroxide, citric acid and potassium hydroxide.

Examples of solvents include, but are not limited to, water,isopropanol, benzyl alcohol, and propylene glycol.

Examples of moisturizing agents include, but are not limited to,glycerin, mineral oil, polyoxyethylene hardened castor oil and Vaseline,propylene glycol; paraffins; waxes, such as, for example, bees wax;polyethylene glycols or mixtures thereof, such as, for example, macrogol(macrogol is a mixture of polyethylene glycols of different molecularweights); stearyl alcohol; benzyl alcohol; parahydrobenzoate esters(parabens); gelled hydrocarbon; citric acid; squalene; lanolins;glycerin; polyoxyethylene hardened castor oil; sorbitan fatty ester;glycerin fatty ester; animal and vegetable fats; oils; starch;tragacanth; cellulose derivatives; silicones; bentonites; silicic acid;talc; zinc oxide and mixtures thereof.

Examples of stabilizers include, but are not limited to, carbohydratessuch as, for example, sucrose, lactose and trehalose; sugar alcoholssuch as, for example, mannitol and sorbitol; amino acids such as, forexample, histidine, glycine, phenylalanine and arginine.

In one embodiment of the invention, the composition, pharmaceuticalcomposition or medicament of the invention may be used in conjunctionwith delivery systems that facilitate delivery of the agents to thecentral nervous system. For example, various blood brain barrier (BBB)permeability enhancers may be used to transiently and reversiblyincrease the permeability of the blood brain barrier to a treatmentagent. Such BBB permeability enhancers include but are not limited toleukotrienes, bradykinin agonists, histamine, tight junction disruptors(e.g., zonulin, zot), hyperosmotic solutions (e.g., mannitol),cytoskeletal contracting agents, and short chain alkylglycerols (e.g.,1-O-pentylglycerol). Oral, sublingual, parenteral, implantation, nasaland inhalational routes can provide delivery of the active agent to thecentral nervous system. In some embodiments, the compounds of thepresent invention may be administered to the central nervous system withminimal effects on the peripheral nervous system.

The blood-brain barrier (BBB) is a physical barrier and system ofcellular transport mechanisms between the blood vessels in the centralnervous system (CNS) and most areas of the CNS itself. The BBB maintainshomeostasis by restricting the entry of potentially harmful chemicalsfrom the blood, and by allowing the entry of essential nutrients.However, the BBB can pose a formidable barrier to delivery ofpharmacological agents to the CNS for treatment of disorders ormaintaining or enhancing normal and desirable brain functions, such ascognition, learning, and memory.

In one embodiment, the inhibitor, the composition, the pharmaceuticalcomposition or the medicament is administered in a sustained-releaseform. In another embodiment, the composition, the pharmaceuticalcomposition or the medicament comprises a delivery system that controlsthe release of the modulator.

In one embodiment, the inhibitor, the composition, the pharmaceuticalcomposition or the medicament of the invention is administered at a dosedetermined by the skilled artisan and personally adapted to eachsubject.

It will be understood that the total daily usage of the inhibitor, thecomposition, pharmaceutical composition and medicament of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective amountfor any particular patient will depend upon a variety of factorsincluding the disease being treated and the severity of the disease; thespecific composition employed, the age, body weight, general health, sexand diet of the subject; the time of administration, route ofadministration, the duration of the treatment; drugs used in combinationor coincidental with the polypeptide or nucleic acid sequence employed;and like factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of a therapeutic compound atlevels lower than those required to achieve the desired therapeuticeffect and to gradually increase the dosage until the desired effect isachieved; but, at the opposite, it can be equally useful to start with aloading dose, a manner to reach steady-state plasma concentration morequickly, and then to follow with a maintenance dose calculated toexactly compensate the effect of the elimination process.

In one embodiment, a therapeutically effective amount of the inhibitor,the composition, the pharmaceutical composition or the medicament of theinvention is administered at least once a day, twice a day, at leastthree times a day.

In another embodiment, a therapeutically effective amount of theinhibitor, the composition, the pharmaceutical composition or themedicament of the invention is administered every two, three, four,five, six days.

In another embodiment, a therapeutically effective amount of theinhibitor, the composition, the pharmaceutical composition or themedicament of the invention is administered twice a week, every week,every two weeks, once a month.

In one embodiment of the invention, the daily amount of the inhibitor,the composition to be administered to a subject ranges from about 2mg/day to about 2000 mg/day, from about 2 mg/day to about 1500 mg/day,from about 2 mg/day to about 1000 mg/day, from about 2 mg/day to about500 mg/day, from about 2 mg/day to about 200 mg/day, from about 5 mg/dayto about 2000 mg/day, from about 5 mg/day to about 1500 mg/day, fromabout 5 mg/day to about 1000 mg/day, from about 5 mg/day to about 500mg/day, from about 5 mg/day to about 200 mg/day, from about 10 mg/day toabout 2000 mg/day, from about 10 mg/day to about 1500 mg/day, from about10 mg/day to about 1000 mg/day, from about 10 mg/day to about 500mg/day, from about 10 mg/day to about 200 mg/day.

In one embodiment of the invention, the daily amount of the inhibitor,the composition to be administered to a subject ranges from about 1mg/kg/day to about 20 mg/kg/day, from about 1 mg/kg/day to about 15mg/kg/day, from about 1 mg/kg/day to about 12 mg/kg/day, from about 1mg/kg/day to about 10 mg/kg/day, from about 1 mg/kg/day to about 9mg/kg/day, from about 1 mg/kg/day to about 8 mg/kg/day, from about 1mg/kg/day to about 7 mg/kg/day.

In another embodiment, the inhibitor, the composition of the inventionis to be administered at a quantity of about 5 mg to about 2000 mg, fromabout 5 mg to about 1500 mg, from about 5 mg to about 1000 mg, fromabout 5 mg to about 500 mg, from about 5 mg to about 200 mg.

In one embodiment, the method of the invention is for a chronictreatment. In another embodiment, the method of the invention is for anacute treatment.

In one embodiment of the invention, the subject is diagnosed with a freeoxygen-radicals related disease. In another embodiment of the invention,the subject is at risk of developing a free oxygen-radicals relateddisease.

In one embodiment, said subject is an adult, a teenager, a child, ayoung child or a new born child.

Another object of the invention is a conservation medium comprising theinhibitor of the invention.

In one embodiment, the conservation medium is for the preservation oforgans. In one embodiment, said organs include, but are not limited to:heart, liver, kidney, lung, pancreas, intestine. In one embodiment, saidorgans are for transplantation.

In one embodiment, the conservation medium comprises the inhibitor ofthe invention at a concentration ranging 5 μM to 120 μM, i.e., at aconcentration of about 5 μM, 10 μM, 20 μM, 50 μM, 80 μM, 100 μM or 120μM.

Another object of the present invention is a method for inhibiting freeoxygen-radicals production in a subject in need thereof by acting onmitochondria at site I_(Q) of complex I, comprising the administrationof an effective amount into the subject of an inhibitor of reactiveoxygen species.

Another object of the present invention is a method for treating agingdiseases in a subject in need thereof by acting on mitochondria at siteI_(Q) of complex I, comprising the administration of an effective amountinto the subject of an inhibitor of reactive oxygen species.

Another object of the present invention is a method for inhibiting freeoxygen-radicals production in a subject in need thereof withoutinhibiting cytosolic ROS production, comprising the administration of aneffective amount into the subject of an inhibitor of reactive oxygenspecies.

Another object of the present invention is a method for increasinginsulin secretion in a subject in need thereof by acting on mitochondriaat site I_(Q) of complex I, comprising administrating an effectiveamount of an inhibitor of production of reactive oxygen species asdescribed here above.

Another object of the present invention is a method for protectingneurons in a subject in need thereof by acting on mitochondria at siteI_(Q) of complex I, comprising administrating an effective amount of aninhibitor of production of reactive oxygen species as described hereabove.

Another object of the invention is an inhibitor of mitochondrialreactive oxygen species production for treating at least one diseaserelated to free oxygen-radicals.

Another object of the invention is the use of an inhibitor ofmitochondrial reactive oxygen species production for treating at leastone disease related to free oxygen-radicals.

Another object of the invention is the use of an inhibitor ofmitochondrial reactive oxygen species production for the preparation ofa medicament for treating at least one disease related to freeoxygen-radicals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A to D) illustrates the absence of effect of AOL onmitochondrial respiration.

FIG. 1A: After incubation in the presence of AOL (20 μM in thisexample), isolated mitochondria from rat heart were oxidizingglutamate+malate (GM) as substrate. Phosphorylation was triggered byadenosine diphosphate (ADP) and stopped by Atractyloside (ATR), aspecific inhibitor of adenine translocator.

FIG. 1B: The classical study of mitochondrial oxidative phosphorylationin the presence of succinate (+ rotenone) was carried out in thepresence of increasing concentrations of AOL (from 5 to 80 μM). Therewere no statistical differences in mitochondrial respiration under thedifferent energetic states after addition of AOL. Oxidation rate afterADP addition reflects the adenosine triphosphate (ATP) synthesisactivity of isolated mitochondria.

FIG. 1C: The classical study of mitochondrial oxidative phosphorylationin the presence of glutamate and malate was carried out in the presenceof increasing concentrations of AOL (from 5 to 80 μM). There were nostatistical differences in mitochondrial respiration under the differentenergetic states after addition of AOL. Oxidation rate after ADPaddition reflects the adenosine triphosphate (ATP) synthesis activity ofisolated mitochondria.

FIG. 1D: The classical study of mitochondrial oxidative phosphorylationin the presence of glutamate, malate and succinate was carried out inthe presence of increasing concentrations of AOL (from 5 to 80 μM).There were no statistical differences in mitochondrial respiration underthe different energetic states after addition of AOL. Oxidation rateafter ADP addition reflects the adenosine triphosphate (ATP) synthesisactivity of isolated mitochondria.

FIG. 2 (A to E) presents the main sites of oxygen radicals' productionby isolated mitochondria in the presence of substrates of both complexesI and III in the presence of ATR (state 4) in order to obtain maximalmitochondrial ROS production. As previously stated, mitochondrial ROSproduction is highly dependent on mitochondrial activity and conditions.Although AOL has been tested under numerous conditions, for the sake ofclarity, we chose to present here only the most demonstrative results.The presence of all the substrates (i.e. Glutamate+Malate+Succinate),giving the electrons to the whole chain, is the closest to in situconditions in the cell. Under these conditions of substrates, weevaluated the effect of the presence of AOL on ROS production by thecomplete chain under ATR (inhibition of phosphorylation: maximumproduction), and by the complex I (inhibited by rotenone) and complex II(inhibited by Antimycin A).

FIG. 2A: production of oxygen radicals by mitochondria in the presenceof malate, glutamate and succinate.

FIG. 2B: production of oxygen radicals by mitochondria in the presenceof rotenone.

FIG. 2C: production of oxygen radicals by mitochondria in the presenceof rotenone and antimycin A.

FIG. 2D: production of oxygen radicals by mitochondria in the presenceof rotenone, antimycin A and myxothiazol.

FIG. 2E: chart illustrating the inhibitor titration design implementedto decipher the action of AOL on ROS production by the whole respiratorychain under conditions of maximal ROS production.

FIG. 3 presents the effect of AOL (5 to 80 μM) on ROS/H₂O₂ production byisolated mitochondria in the presence of substrates of complexes I andII in the presence of ATR (state 4), effect of rotenone, antimycin A andmyxothiazol. In the absence of specific inhibitors of the complexes, ROSproduction is at maximum and mainly comes from reverse electrontransport at site I_(Q) (see FIG. 4). After addition of rotenone, whichspecifically reverse electron transport by inhibiting I_(Q), productiondecreases and occurs almost entirely at site III_(QO). The subsequentaddition of Antimycin A, which blocks the transfer of the electrons tooxygen, increases ROS production at site III_(QO) and finallymyxothiazol blocks ROS production at site III_(QO) (see FIG. 2 fordetails).

FIG. 4 is a scheme presenting the site of action of AOL on mitochondriaROS production and the sites where AOL has no or little action.

FIG. 5 is a histogram showing the effect of 10 and 20 μM AOL on glucosestimulated insulin secretion (GSIS) in isolated islets from maleC57Bl/6J mice. Combination of three experiments is displayed. Isletsfrom two mice for each experiment; five islets each well; four to sixwells each condition. Insulin secretion data were normalized to 11 mMGlc-Veh group, which was considered 100%. *p<0.05, **p<0.01 and***p<0.001 versus 3 mM Glc-Veh; # p<0.05, ## p<0.01 and ### p<0.001versus 11 mM Glc-Veh; One-way ANOVA and Bonferroni's post-hoc test.

FIG. 6 is a histogram showing the fat mass determined after 3 weeks oftreatment. Fat mass is expressed in grams (g). Data are expressed asmean±SEM.

FIG. 7 is a histogram showing the lean mass determined after 3 weeks oftreatment. Lean mass is expressed in grams (g). Data are expressed asmean±SEM.

FIG. 8 is a graph showing the effect of chronic treatment with AOL (5mg/kg and 10 mg/kg) on glucose responses during an insulin tolerancetest (ITT). The graph represents changes in blood glucose levels duringan ITT. Data are expressed as mean±SEM.

FIG. 9 is a histogram showing the effect of AOL on blood glucose levels.After five weeks of treatment, blood glucose was measured in 2-h fastedmice. Data are expressed as mean±SEM.

FIG. 10 is a histogram showing the neuroprotective effect of AOL (5mg/kg bid for 11 days) on TH-positive cell counts in the SN inMPTP-treated mice. Data expressed as mean±SEM (n=10-11) and analyzedusing one-way repeated measures ANOVA followed by Dunnett's multiplecomparison test. **P<0.01;***P<0.001 c.f. MPTP+vehicle.

FIG. 11 is a graph showing the effect of AOL on the recovery of heartcontractility during the reperfusion phase following ischaemia. Data areexpressed as mean±SEM for control (black) and AOL treated (grey) for 6independent experiments.

FIG. 12 is a graph showing the effect of AOL on the infarct size of theslices of ischaemic hearts. At the end of the reperfusion period, heartswere stained by triphenyltetrazolium chloride (TTC). Living tissueappears red, while damaged tissue appears white.

FIG. 13 (A and B) is a set of two graphs showing the effect of AOLtreatment on pulmonary arterial pressure and heart remodeling.

FIG. 13A: effect of AOL (hatched columns) on the mean pulmonary arterialpressure (mPAP) measured in normoxic rats (N, white columns), chronichypoxic rats (CH, light grey columns) and monocrotaline-treated rats(MCT, dark grey columns) n is the number of rats. *, ** and *** indicatea significant difference for P<0.05, 0.01 and 0.0001 respectively versusN. ### indicates a significant difference for P<0.05 versus CH. † and ††indicate a significant difference for P<0.05 and 0.01 respectivelyversus N+AOL. ‡ indicates a significant difference for P<0.05 versusMCT.

FIG. 13B: right ventricular hypertrophy expressed as the Fulton index(i.e., ratio of right ventricle weight (RV) to left ventricle plusseptum weight (LV+S)). n is the number of rats. *, ** and *** indicate asignificant difference for P<0.05, 0.01 and 0.0001 respectively versusN. ### indicates a significant difference for P<0.05 versus CH. † and ††indicate a significant difference for P<0.05 and 0.01 respectivelyversus N+AOL. ‡ indicates a significant difference for P<0.05 versusMCT.

FIG. 14 (A to C) is a set of graphs showing the effect of AOL onpulmonary arteries (PA) remodeling. The effect of AOL (hatched columns)on PA remodeling was assessed by measuring the percentage of PA medialthickness in normoxic rats (N, white columns), chronic hypoxic rats (CH,light grey columns) and monocrotaline-treated rats (MCT, dark greycolumns). Intraacinar arteries observed to estimate PA remodeling wereseparated into three groups with different cross-sectional diameters.

FIG. 14A: PA medial thickness assessed in rats with intraacinar arteriesof cross-sectional diameter under 50 n is the number of vessels. *, **and *** indicate a significant difference for P<0.05, 0.01 and 0.0001respectively versus N. ## and ### indicate a significant difference forP<0.01 and 0.0001 versus CH. a significant difference for P<0.05 and0.01 respectively versus N+AOL. ‡ indicates a significant difference forP<0.05 versus MCT.

FIG. 14B: PA medial thickness assessed in rats with intraacinar arteriesof cross-sectional diameter between 50 to 100 μm. n is the number ofvessels. *, ** and *** indicate a significant difference for P<0.05,0.01 and 0.0001 respectively versus N. ## and ### indicate a significantdifference for P<0.01 and 0.0001 versus CH. a significant difference forP<0.05 and 0.01 respectively versus N+AOL. ‡ indicates a significantdifference for P<0.05 versus MCT.

FIG. 14C: PA medial thickness assessed in rats with intraacinar arteriesof cross-sectional diameter between 100 to 150 n is the number ofvessels. *, ** and *** indicate a significant difference for P<0.05,0.01 and 0.0001 respectively versus N. ## and ### indicate a significantdifference for P<0.01 and 0.0001 versus CH. a significant difference forP<0.05 and 0.01 respectively versus N+AOL. ‡ indicates a significantdifference for P<0.05 versus MCT.

FIG. 15 (A and B) is a set of graphs showing the effect of AOL on thethickness of the outer nuclear layer (ONL) of the retina, in progressivelight-induced retinal degeneration.

FIG. 15A: effect of vehicle and AOL on “untransferred” animals. Animalswere bred under cyclic low-intensity lighting and received injections ofvehicle or AOL three times a day for 7 days. Fifteen days after the endof the treatment, histological analysis of the retina was carried out.Data are expressed as mean±SEM thickness of the ONL, for untreatedanimals (light grey, square dots), vehicle-treated animals (dark grey,triangle dots) and AOL-treated animals (black, round dots), in μm, fromthe optical nerve and every 0.39 mm in the superior and inferior polesof the optic disc.

FIG. 15B: effect of vehicle and AOL on “transferred” animals. Animalswere bred under cyclic low-intensity lighting and transferred to cyclichigh-intensity lightning for 7 days, during which they receivedinjections of vehicle or AOL three times a day. At the end of thetreatment, animals were transferred back under cyclic low-intensitylighting conditions, and histological analysis of the retina was carriedout fifteen days later. Data are expressed as mean±SEM thickness of theONL, for untreated animals (light grey, square dots), vehicle-treatedanimals (dark grey, triangle dots) and AOL-treated animals (black, rounddots), in μm, from the optical nerve and every 0.39 mm in the superiorand inferior poles of the optic disc.

FIG. 16 (A to C) is a set of graphs showing a lifespan SOD2-KOexperiment on four groups of mice (WT-KOL: wild-type mice treated withvehicle; WT-AOL: wild-type mice treated with AOL; KO-KOL: SOD2-KO micetreated with vehicle; KO-AOL: SOD2-KO mice treated with AOL). Data areexpressed as mean values.

FIG. 16A: evolution of mice body weight, in grams over time, in days.

FIG. 16B: baseline-corrected of mice body weight, as a percentage ofweight gain over time, in days.

FIG. 16C: survival proportion among the KO-KOL and KO-AOL groups, inpercentage over time, in days.

FIG. 17 is a graph showing the succinate dehydrogenase (SDH) activity inheart, among five groups of mice (WT-KOL: wild-type mice treated withvehicle; WT-AOL: wild-type mice treated with AOL; KO-KOL: SOD2-KO micetreated with vehicle; KO-AOL: SOD2-KO mice treated with AOL; WT:wild-type untreated mice). The optical density of SDH reaction sampledfrom heart sections was measured with the image processing softwareImage Analyst MKII (Akos). This density was expressed in the form ofmean grey level where mean grey level=sum of grey/number of pixelsmeasured. Data are expressed as mean values of the measured opticaldensity.

FIG. 18 (A to C) is a set of graphs showing Oil Red O staining of liverslices from WT and SOD2-KO mice, treated or not with AOL.

FIG. 18A: Average size of lipids droplets.

FIG. 18B: droplet density (droplets number/liver area).

FIG. 18C: total lipid area (average size×droplets number).

EXAMPLES

The present invention is further illustrated by the following examples.

Example 1: AOL does not Affect Mitochondrial Oxidative Phosphorylation

Material and Methods

Animal Procedures and Ethics Statement

All experiments described were carried out in agreement with theNational and European Research Council Guide for the care and use oflaboratory animals. P. Diolez has a valid license to conduct experimentson animals by the Service Vétérinaire de la Santé et de la ProtectionAnimale of the Ministère de 1'agriculture et de la Forêt, France(03/17/1999, license number 3308010).

Materials

All the chemicals were reagent grade, purchased from Sigma Chemical (St.Louis, Mo.), except for sucrose and NADH oxidase (that were obtainedfrom Merck (Darmstadt, Germany)). The trithio-AnethOL compound (AOL) wasa gift from the private company GMPO (Paris, France). 15 mM stocksolution was prepared in DMSO, and kept in darkness at 0° C. for onlyfew days.

Isolation of Mitochondria

Male Wistar rats (250-325 g; obtained from Janvier Labs, LeGenest-Saint-Isle, France) were killed by stunning and cervicaldislocation, and the heart was quickly removed and washed in coldisolation medium containing 100 mM sucrose, 180 mM KCl, 50 mM Tris, 5 mMMgCl₂, 10 mM EDTA, and 0.1% (w/v) deffated BSA (pH 7.2).

Isolation of heart mitochondria was performed in a cold chamber. Beforehomogenization, hearts (about 1.5 g) were minced with scissors andtreated for 5 min in 5 mL of the same medium supplemented with protease(2 mg of bacterial proteinase type XXIV per mL of isolation buffer) withstirring. The tissue suspension was poured into a 50-ml glass Potterhomogenizer, diluted with 20 mL of isolation buffer, then homogenizedfor 3 min using a motorized Teflon pestle. The homogenate was filteredthrough bolting cloth (Sefar Nitex) to remove debris, and centrifuged at8,000 g for 10 minutes. The resulting pellet was rinsed with 5 mL ofisolation buffer, resuspended in 25 mL of the same buffer, thensubjected to low speed centrifugation (400 g) for 8 mM. The resultingsupernatant was centrifuged twice at 7,000 g for 15 min to yield awashed mitochondrial pellet that was gently resuspended in 150 μL ofisolation buffer. Protein concentration was determined by the Bradfordmethod (Sigma, kit # B6916) using BSA as standard. Mitochondria werekept on ice at a final concentration of 40-50 mg/mL for less than 5 h.

Mitochondrial Respiration

Oxygen consumption rates of heart mitochondria (0.1 mg/mL), incubated inthe absence or presence of AOL at increasing doses (from 0 to 80 μMfinal concentration), were recorded polarographically under constantstirring at 25° C. using a high resolution oximeter (Oxygraph-2K,Oroboros Instruments, Austria). The respiration medium consisted in 140mM sucrose, 100 mM KCl, 1 mM EGTA, 20 mM MgCl₂, 10 mM KH₂PO₄, and 1 g/L(w/v) BSA essentially fatty acid free (pH 7.2).

Mitochondrial, ROS/H₂O₂, Production

Rates of ROS/H₂O₂ production from heart mitochondria were assessedthrough the oxidation of the colorless, non-fluorescent indicator AmplexRed in the presence of exogenous horseradish peroxidase (HRP, EC1.11.1.7, Sigma). H₂O₂ reacts with Amplex Red in a 1:1 stoichiometry,yielding the fluorescent compound resorufin (excitation: 560 nm;emission: 585 nm) which is stable once formed. Fluorescence was measuredcontinuously with a spectro-fluorometer equipped with temperaturecontrol and stirring (SAFAS Xenius, Monaco). Isolated mitochondria (0.1mg/mL) were incubated in the same experimental buffer than previously,supplemented with 15 μM Amplex Red and 10 μg/mL HRP. Glutamate (5mM)/malate (2.5 mM) together with succinate (5 mM) were used as complexI and complex II substrates, respectively. Experiments were conductedunder non-phosphorylating conditions in the presence of 15 μMatractyloside, i.e. under state IV conditions where mitochondrialmembrane is maximal. Afterwards, rotenone (1.5 μM), antimycin A (2 μM),and myxothiazol (0.2 μM) were sequentially added to inhibit the redoxcenters within the electron transfer chain (see FIG. 2), namely sitesI_(Q), I_(F) (with rotenone), III_(Q), (with antimycin A) and III_(QO)(with myxothiazol). Assay was finally calibrated with known amounts ofH₂O₂ (steps of 300 nM), in the presence of all relevant compounds,including AOL. The control test of the absence of effect of AOL on theamplex red assay itself and NAD(P)H oxidase ROS/H₂O₂ production wascarried out in the absence of cardiac mitochondria and the presence ofNAD(P)H oxidase (EC 1.6.3.3, 5 mU/mL, Sigma) and NADH (100 μM)solutions.

Results

We first verified (FIG. 1) that the AOL compound did not affectoxidative phosphorylation directly on isolated mitochondria from ratheart. This has been carried out by using the now classical oxygraphmethod. Mitochondria were first incubated with various AOLconcentrations (5 to 80 μM) then respiratory substrate was added(substrate state, black curve), followed by a saturating ADPconcentration to get the maximal oxidative phosphorylation rate (greycurve), and finally the addition of atractyloside (ATR) which inhibitsthe ADP/ATP translocator and gives the mitochondrial leak rate undernon-phosphorylating conditions (FIG. 1A). The other panels of FIG. 1B-Dpresents the results obtained with different respiratory substratecombinations: glutamate+malate which feed electrons to complex I,succinate (+ rotenone) for complex II and glutamate+malate+succinate tofeed electrons to both complexes. This last substrate combination hasbeen chosen since it most closely resembles to in vivo conditions whereKrebs cycle functions and both succinate and NADH are oxidized byrespiratory chain. The results indicate that statistically nodifferences were observed in the presence of AOL for the large range ofconcentrations tested (FIG. 1), demonstrating under these conditions theabsence of effect of AOL on mitochondrial oxidative phosphorylation—i.e.both on respiratory chain activity and ATP synthesis—as well as onmitochondrial inner membrane integrity (leak rate, after ATR addition).This last result indicates that AOL does not affect oxidativephosphorylation yield. Together, all these results confirm the absenceof any harmful effect of AOL, documented by the use of this drug forhuman health for a long time.

Example 2: AOL Inhibits Superoxide/H₂O₂ Production by Mitochondria

As previously stated, mitochondrial ROS production is highly dependenton mitochondrial activity and conditions. Although we tested the effectsof AOL on ROS production by mitochondria under numerous conditions, wechose to present here, for the sake of clarity, only the mostdemonstrative results of the very specific effects of AOL. As alreadydiscussed, the presence of the substrate combination (i.e.Glutamate+Malate+Succinate), giving the electrons to the wholerespiratory chain, is the most representative of in situ conditions inthe cell where the metabolism is active. Furthermore, maximalmitochondrial ROS/H₂O₂ production does not occur under conditions ofhigh mitochondrial phosphorylation but under conditions of highreduction of electron transporters, i.e. low or no phosphorylation.These conditions are fulfilled in the presence of ATR (inhibition ofATP/ADP translocator by ATR, (FIG. 1) and we could effectively verifythat the addition of ATR under conditions of saturating ADP triggeredthe production of ROS which was at the detection limit under maximalphosphorylating conditions (results not shown). Under these conditions,ROS are produced at different sites of the respiratory chain (Orr etal., 2013. Free Radic. Biol. Med. 65:1047-1059; Quinlan et al., 2013.Redox Biol. 1:304-312) (FIG. 2). The main sites of production arelocated at complexes I and III, where large changes in potential energyof electrons occur (Balaban et al., 2005. Cell. 120:483-495; Goncalveset al., 2015 Jan. 2. J. Biol. Chem. 290(1):209-27), which also allowproton pumping at these sites.

We designed a series of inhibitor titrations in order to decipher theaction of AOL on ROS production by the whole respiratory chain underconditions of maximal ROS production (FIG. 2E). In the absence ofspecific inhibitors of the complexes, ROS production is at maximum andmainly comes from reverse electron transport at site I_(Q) (FIG. 2A). Itis crucial to note that ROS produced by complex I, either by site I_(Q)(quinone site) or site I_(f) (flavin site), are delivered to theinner—matrix—side of the inner mitochondrial membrane. After addition ofrotenone, a classical inhibitor of complex I which specifically binds toI_(Q), the ROS production decreases strongly and occurs almost entirelyat site III_(QO) and a remaining production at site I_(f) due to thepresence of complex I substrates and NADH production which are notinhibited by rotenone (FIG. 2B). The subsequent addition of antimycin A,an inhibitor of the electron transfer to cytochrome c, causes anincrease in the reduced over oxidized quinone ratio, which is stillreduced by complex II activity, and therefore a concomitant increase inROS production at site III_(QO) (FIG. 1C). Finally, the addition ofmyxothiazol, an inhibitor of complex III site III_(QO), abolishescomplex III ROS production and the remaining very low production may beascribed to the flavin site of complex I, for which we have no knowninhibitor (Goncalves et al., 2015 Jan. 2. J. Biol. Chem. 290(1):209-27).

FIG. 3 illustrates the effect of the presence of increasingconcentrations of AOL (from 5 to 80 μM) on ROS/H₂O₂ production measuredunder the different conditions defined in FIG. 2. It clearly appearsfrom the results presented in this figure that AOL only affects the ROSproduction measured in the absence of inhibitor, by approximately 80%,while no statistical differences were observed with this range of AOLconcentrations on ROS/H₂O₂ measured under the other conditions. As canbe seen on FIG. 2, this specific condition (only ATR present) is theonly condition in our assay where ROS are produced by complex I (siteI_(Q)). When rotenone is added to the assay, ROS/H₂O₂ production appearsinsensitive to AOL, even at high concentrations, whatever the site beinginvolved. The clear absence of effect on several sites of mitochondrialROS production is not only surprising but also asks interestingquestions about the very mechanism of action of AOL on mitochondria.Indeed, these results rule out the basic hypothesis of the mode ofaction of AOL described in the previous papers and at the basis of thepatent for its therapeutic use. These results effectively demonstratethat AOL is not a radical scavenger; otherwise its action would beindependent of the origin of the ROS. However, since AOL clearlystrongly decreases ROS production by complex I at site I_(Q), and onlythat site, we have the evidence that AOL specifically inhibits theformation of ROS at this site.

Although the mechanism has still to be investigated, evidence ispresented here that AOL compound specifically interferes withmitochondrial complex I and selectively inhibits superoxide productionfrom the ubiquinone-binding site of complex I (site I_(Q)) with noeffects on superoxide production from other sites or on oxidativephosphorylation. To our knowledge, there is only one compound withcomparable properties that has recently been described, theN-cyclohexyl-4-(4-nitrophenoxy) benzenesulfonamide (Orr et al., 2013.Free Radic. Biol. Med. 65:1047-1059). Like AOL, this compound does notmodify the activity of complex I as a component of the respiratory chainand oxidative phosphorylation.

The specificity of AOL was further tested in vitro using theperoxidase-Amplex red system utilized for the measurement of ROS/H₂O₂ bymitochondria, which in fact measures the appearance of H₂O₂ by theoxidation of amplex red to the fluorescent resorufin (see FIG. 4). Inthe absence of mitochondria and by adding instead a H₂O₂-producingsystem to the measuring system, it was possible to test the effect ofAOL on this system. This has been carried out by using commercialNAD(P)H oxidase which produces H₂O₂ in the presence of added NAD(P)H andmeasuring the reduction of Amplex red to resorufin (FIG. 4). We did notobserve any inhibition of the fluorescence under these conditions, whichexclude any effect of AOL on the NAD(P)H oxidase or on the peroxidaseactivity (results not shown). These results confirm that AOL does notinterfere either with the measurement system or directly interact withH₂O₂. Interestingly, these results also demonstrate that AOL does notinhibit the ROS/H₂O₂ production by the NADP(H) oxidase, which is one—ifnot the—major non-mitochondrial ROS/H₂O₂ producer in the cells. Thescheme on FIG. 4 recapitulates the different informations on the mode ofaction of AOL on ROS/H₂O₂ production by mitochondria and NAD(P)H oxidaseand stresses the very high specificity demonstrated here. These resultsare in striking contrast with previous assertions on the putative effectof AOL as radical scavenger.

When tested on isolated mitochondria from rat heart, AOL effectivelydecreases mitochondrial ROS/H₂O₂ production (in isolated mitochondria,H₂O₂ is produced from the reduction of ROS by mitochondrial superoxidedismutase). However, the results presented here clearly demonstrate thatAOL does not act as a simple antioxidant or radical scavenger. Whileantioxidants are general ROS/H₂O₂ scavengers, AOL presents a completeselectivity towards the formation of ROS by site I_(Q) in complex I,which demonstrates that AOL does not simply interact with superoxideradicals but specifically prevents their formation in complex I. In thatrespect, AOL therefore appears as a member of a brand new class ofoxidative stress protectants, whose only one member has been describedvery recently (Orr et al., 2013. Free Radic. Biol. Med. 65:1047-1059).Whereas antioxidants generally do not interfere directly with electrontransport and scavenge ROS and/or H₂O₂ downstream from production andtherefore can never fully suppress the effect of ROS (Orr et al., 2013.Free Radic. Biol. Med. 65:1047-1059), AOL may act differently bypreventing ROS formation and thus being more active to protectmitochondria from their own ROS.

Data presented here go further and demonstrate that AOL is a specificinhibitor of ROS formation at site I_(Q) of complex I of mitochondrialrespiratory chain. Further experiments are however required to ascertainthat AOL has completely no effect on other mitochondrial sites, but thisdoes not preclude the above conclusions. We also show here someevidences that AOL may only interact with mitochondria without affectingoxygen radicals formation in cytosol, and therefore would not affectintracellular signalisation.

Inhibition of complex I activity by rotenone or the neurotoxin MPP hasbeen linked to parkinsonism in both rodents and humans, suggesting alink between dysfunctional complex I, ROS production, andneurodegeneration (Langston et al., 1983. Science. 219:979-980; Betarbetet al., 2000. Nat. Neurosci. 3:1301-1306). In contrast, comparativeanalyses show an inverse relationship between maximal superoxide/H₂O₂production from site I_(Q), but not site I_(F), and maximum life spanacross diverse vertebrate species (Lambert et al., 2007. Aging Cell.6:607-618; Lambert et al. 2010. Aging Cell. 9:78-91). Therefore,selective modulators of superoxide/H₂O₂ production from site I_(Q) orsite I_(F) would offer unique opportunities to probe the putative roleof mitochondrial ROS production in normal and pathological processes(Orr et al., 2013. Free Radic. Biol. Med. 65:1047-1059). There are alsosome speculations, even controversial, that site III_(QO)—not affectedby AOL—play an important role in cellular signalling during hypoxia.

In conclusion, it appears that AOL properties may represent abreakthrough in the search for specific modulators of ROS/H₂O₂production in cells. This is a current important issue in research, andAOL has an enormous advantage toward newly discovered molecules since itis already authorized for human use.

-   -   AOL acts upstream from ROS production, therefore insuring higher        protection than classical antioxidants;    -   AOL acts specifically on mitochondrial ROS production;    -   AOL ensures mitochondrial protection, crucial for numerous        diseases, especially cardiac ones;    -   AOL does not interfere with cell signalisation;    -   AOL acts specifically on site I_(Q) in complex I, which is the        main mitochondrial site and may be implicated in important        diseases, including Parkinson's disease and cardiac        fibrillation.

→AOL may represent the first member of a new class of “protectants” thatspecifically prevent ROS production inside mitochondria, and maytherefore be used for mitochondrial protection during various oxidativestress and therefore prevent diseases, with very little side effects oncrucial cellular ROS signalling.

Example 3: Effect of AOL in a Cardiovascular Disease: Diabetes

Effect of the Compound AOL on Glucose-Stimulated Insulin Secretion(GSIS) in Mouse Pancreatic Islets

The aim of the study was to investigate the ability of the compound AOLin modulating glucose-stimulated insulin secretion (GSIS) in isolatedpancreatic islets from mice.

Material and Methods

Experiments were conducted in strict compliance with the European Unionrecommendations (2010/63/EU) and were approved by the French Ministry ofAgriculture and Fisheries (authorization n° 3309004) and the localethical committee of the University of Bordeaux. Maximal efforts weremade to reduce the suffering and the number of animals used.

Three independent experiments were carried out and, for each of them,two mice were sacrificed and islets isolated according to the procedurefurther described below.

Pancreatic islets were isolated using the collagenase digestion method.Briefly, pancreas was inflated with Hanks solution containing 0.33 mg/mLof collagenase (Sigma-Aldrich), 5.6 mM glucose and 1% bovine serumalbumin, pH 7.35, removed and kept at 37° C. for 6-9 minutes. Aftertissue digestion and exocrine removal by three consecutive washes, theislets were manually collected, under a binocular magnifier. Islets wereleft recovering from digestion by culturing for 20-24 hours in RPMI-1640medium containing 11 mM glucose (Invitrogen, Calif., USA) andsupplemented with 2 mM glutamine, 200 IU/mL penicillin, 200 μg/mLstreptomycin and 8% fetal bovine serum stripped with charcoal-dextran(Invitrogen).

For each static GSIS experiment, islets from two mice were firstincubated for 2 hours at 37° C. in 3 mL Krebs-bicarbonate buffersolution (in mM): 14 NaCl, 0.45 KCl, 0.25 CaCl₂, 0.1 MgCl₂, 2 HEPES and3 glucose, equilibrated with a mixture of 95% 02:5% CO₂, pH 7.4. Then,groups of five size-matched islets were transferred to 24-well platewells with 0.5 mL fresh buffer containing either one of the followingstimulus: 3 mM glucose (Glc) and 11 mM glucose plus vehicle (0.4% DMSOin Krebs-bicarbonate buffer), or 11 mM glucose plus the diluted drug tobe tested (10 μM or 20 μM of AOL in vehicle), and further incubated for1 hour. Six different wells were used for each experimental condition.At the end of the incubation, bovine albumin was added to each well to afinal concentration of 1%, and the plate was put at 4° C. for 15 minutesto stop insulin secretion. Next, the media was collected and stored at−20° C. for subsequent measurement of insulin content by ELISA (kit fromMercodia, Uppsala, Sweden), according to the manufacturer'sinstructions. Insulin secretion in each well was calculated as ng ofinsulin per islet and per hour of incubation, and then expressed aspercentage of insulin secretion in 11 mM glucose vehicle group, whichwas considered 100%.

Description of the experimental groups is shown in Table 1.

TABLE 1 Group abbreviation 3 mM Glc- 11 mM Glc- 11 mM Glc + 11 mM Glc +Veh Veh AOL 10 μM AOL 20 μM Group Group Group Group treated Grouptreated definition treated treated with AOL with AOL with vehicle withvehicle 10 μM 20 μM and 3 mM and 11 mM and 11 mM and 11 mM glucoseglucose glucose glucose Number of 4-6 5-6 4-6 6 wells

Results

Individual insulin secretion values obtained in each of the threeexperiments were combined and averaged. These are expressed as therelative percentage of insulin secretion, normalized to the 11 mMglucose vehicle group (FIG. 5).

Combined analysis of data shows that AOL enhanced GSIS at both 10 and 20μM, showing a similar potency, with a GSIS increase ranging around65-75% as compared to the 11 mM glucose vehicle group (One-way ANOVA;Bonferroni's post-test). For statistical analysis see Table 2.

TABLE 2 Degrees One-way Sum of of Mean of ANOVA squares freedom squaresF P Experiment Treatment 50630 3 16880 18.36 <0.0001 #1 Residual 1654018 919.0 Experiment Treatment 44790 3 14930 21.46 <0.0001 #2 Residual12530 18 695.9 Experiment Treatment 193700 3 64570 35.16 <0.0001 #3Residual 33060 18 1837 Combined Treatment 244100 3 81360 33.13 <0.0001experiments Residual 152200 62 2455

Conclusion

The study demonstrates that AOL, at the doses tested (10 and 20 μM),enhances GSIS and significantly stimulates insulin secretion in vitro,in isolated pancreatic islets from mice.

Thus, these findings suggest that AOL might be particularly useful inpathological conditions in which insulin secretion is deficient.

Effects of a Chronic Treatment with AOL on Food Intake, Body Weight andGlucose Metabolism in Diet-Induced Obese Mice

Material and Methods

The aim of the study was to determine whether the compound AOL,administered daily at the doses of 5 mg/kg and 10 mg/kg for up to fiveweeks by intraperitoneal (ip) daily administration in diet-induced obese(DIO) mice fed with a high-fat diet (HFD), modifies food intake, bodyweight, adiposity and glucose metabolism.

Mice were fed ad libitum with a HFD (60% of calories from fat, mostlylard) for twelve weeks before the pharmacological study begun. Animalsreceived AOL or its vehicle by intraperitoneal (ip) administration andwere maintained on HFD for the length of the study. Food intake and bodyweight were measured daily and recorded for up to three consecutiveweeks.

For appropriate distribution of the mice in the different experimentalgroups before the start of the pharmacological study, we evaluated theirbody composition in vivo using an Echo MRI 900 (EchoMedical Systems,Houston, Tex., USA) (see also Cardinal P. et al., 2014 October Mol.Metab. 3(7):705-16; Cardinal P. et al., 2015 February Endocrinology.156(2):411-8). Daily food intake and body weight measurements wereobtained using a balance (model TP1502, Denver Instruments).

Thirty 7-weeks-old male C57/Bl6J mice arrived to the laboratory on 25Feb. 2016 and underwent a first in vivo body composition analysis (EchoMRI 900, EchoMRI Systems) after 1 week of adaptation to the experimentalhousing room. After this first MRI analysis, animals were fed a high-fatdiet (HFD) ad libitum for a period of twelve weeks. Thereafter, theyunderwent a second MRI analysis and were distributed into 3 experimentalgroups of equivalent body weight and body composition.

Once the pharmacological treatment started (day 1), food intake (FI) andbody weight (BW) were measured daily before the dark phase in animalshoused in their home cage. Spillage of food was checked daily. The foodconsumed was calculated by subtracting the food left in the hoppers fromthe initial pre-weighted amount. H and BW were measured for threeconsecutive weeks. Afterwards animals underwent a third MRI analysis inorder to observe potential effects of the treatment onto the bodycomposition (changes in fat and lean mass), followed by a glucosetolerance test (GTT) and an insulin tolerance test (ITT). Mice receiveddaily ip administration of AOL or its vehicle for a total length of fiveweeks, until they were sacrificed.

A nuclear echo magnetic resonance imaging whole-body compositionanalyzer (Echo MRI 900; EchoMedical Systems) was used to repeatedlyassess body fat and lean mass in conscious mice.

GTT and ITT are routinely used to assess dynamic modulation of glucosemetabolism respectively during a glucose challenge and an insulinchallenge. They give information on the presence of glucose intoleranceand possible resistance to the action of the hormone insulin.

Animals were injected ip with 1.5 g/kg of D-Glucose (Sigma-Aldrich) forthe GTT or with 0.5 U/kg of insulin (Humulin, Lilly, France) for theITT. For the GTT and the ITT, animals were fasted overnight. The testswere conducted the following morning. Blood samples were taken from thetail vein at different time points (0, 15, 30, 60, 90 and 120 minutesafter the ip administration of glucose or insulin) and glucoseconcentration was measured using glucose sticks (OneTouch Vita, LifescanFrance, Issy les Moulineaux, France).

At sacrifice, blood samples were collected, blood glucose was rapidlyassessed using glucose sticks and blood samples were then centrifuged at3000 rpm for 15 minutes. The obtained plasma was stored at −80° C. forsubsequent measurement of insulin, which was carried out by performingan ELISA (kit from Mercodia, Uppsala, Sweden), according to themanufacturer's instructions.

HOMA-IR index, which gives information about the presence of insulinresistance, was calculated using the formula (Glucose mmol/L×InsulinmU/L)/22.5.

Statistical analyses were carried out using GraphPad Prism Software (SanDiego, Calif., USA). Repeated measurements two-way ANOVA were carriedout to analyze the effects of the treatment factor, the time factor andtheir interaction on food intake, body weight, GTT and ITT. One-wayANOVA was carried out to compare the effect of the treatment factor oncumulative food intake, body composition, AUC of GTT and ITT, andcirculating glucose, insulin and HOMA-IR at time of sacrifice. WhenANOVA results were significant (p<0.05), the Tukey post-hoc test wasperformed to allow adequate multiple comparisons among the groups. Dataare expressed as mean±SEM. Graphs were generated using GraphPad Prismsoftware.

Results

The treatment did not have a significant effect on body weight or on thepercentage (%) of change of the body weight calculated from day 1 inwhich body weight was measured before the first administration of AOL.

Chronic administration of AOL after three weeks tended to reduce fatmass (p=0.13, FIG. 6), whilst it did not have any effect on lean mass(FIG. 7). The mean±SEM values are represented in FIG. 6 and FIG. 7 andstatistical analysis are shown in Table 3 and Table 4, respectively.

TABLE 3 Statistical analysis of data represented in FIG. 6. One-way Sumof Degrees of Mean of ANOVA squares freedom squares F p Treatment 18.502 9.248 2.151 0.1366 Residual 111.8 26 4.299

TABLE 4 Statistical analysis of data represented in FIG. 7. One-way Sumof Degrees of Mean of ANOVA squares freedom squares F p Treatment 2.7732 1.387 1.256 0.3014 Residual 28.70 26 1.104

AOL at the dose of 10 mg/kg significantly blunted the action of insulinon circulating glucose levels during an ITT (FIG. 8), suggesting thepresence of insulin resistance. Accordingly, a treatment effect was alsofound when analyzing the AUC (AUC veh: 12812.50±750.35, AUC AOL 5 mg/kg:15006.56±1139.69, AUC AOL 10 mg/kg: 18168.33±1562.90, one-way ANOVA F(2,23)=5.186, p=0.0138), with the AOL 10 mg/kg group having an AUCsignificantly higher than the vehicle group (Tukey post-hoc, p=0.0107).The mean±SEM values are represented in FIG. 8 and statistical analysisare shown in Table 5.

TABLE 5 Post-hoc analysis on the treatment factor for data in FIG. 8.Tukey post-hoc Vehicle AOL 5 mg/kg AOL 10 mg/kg Vehicle 0.5320160.023546 OP 5 mg/kg 0.532016 0.232976 OP 10 mg/kg 0.023546 0.232976 Thenumbers in the Tukey Post-hoc analysis table represent the p values.Values in bold correspond to significant (p < 0.05)results.

At time of sacrifice, after five weeks of treatment, blood glucoselevels were measured in 2-hour fasted mice.

AOL tended to decrease blood glucose levels (FIG. 9) and statisticalanalysis in Table 6.

TABLE 6 Statistical analysis of data represented in FIG. 9. One-way Sumof Degrees of Mean of ANOVA squares freedom squares F p Treatment 3972 21986 2.586 0.0980 Residual 16898 22 768.1

Conclusion

In diet-induced obese animals, chronic daily administration of AOLtended to decrease body weight and food intake in DIO mice (data notshown). Accordingly, this was associated with a trend to decrease fatmass and basal blood glucose levels.

Overall, these data suggest that AOL might have some beneficial effectsin a model of dietary obesity.

Example 4: Effect of AOL in a Neurologic Disease: Parkinson Disease

In this study, the potential neuroprotective effects of AOL wereassessed by counting the number of tyrosine hydroxylase (TH)-positiveneurons in the substantia nigra (SN) in the sub-chronic1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model ofParkinson's disease. Mice were treated with AOL (5 mg/kg; ip) or vehiclefor 11 consecutive days. MPTP (20 mg/kg; ip) or saline was administeredon treatment days 4-8. All mice were killed on day 12 following finaladministration of treatment.

Sub-chronic MPTP administration in C57/bl6 mice induces degeneration ofnigrostriatal dopaminergic neurons, which leads to reduced number ofTH-positive neurons in the SN which was, on this occasion, a reductionof 39%.

Material and Methods

For vehicle conditions, the test item was dissolved in 0.5% DMSO/0.95%Tween 20 in saline while AOL was administered intraperitoneally (i.p.)at the dose of 5 mg/kg. The volume of administration was 10 mL/kg.

C57bl/6 male mice (Janvier) weighing 22-28 g were housed in atemperature-controlled room under a 12-hour light/dark cycle with freeaccess to food and water. In order to tentatively achieve final numbersof n=10 per group, n=12 per group were be used to account for possiblelosses in the course of the experiment. To produce neurodegeneration ofdopaminergic neurons in the substantia nigra, mice were treated withMPTP hydrochloride (20 mg/kg i.p. once daily for five consecutive days).

Mice were humanely euthanized by cervical dislocation after the lastadministration.

The caudal half of the brain (containing the substantia nigra) wasplaced in paraformaldehyde (4% in 0.1 M Phosphate Buffer Saline (PBS) pH7.4) for 5 days and then transferred to 20% sucrose (20% in 0.1 M PBS)for cryoprotection. The tissue was then frozen in cold isopentane (at−50° C. plus or minus 2° C.).

The striata were dissected out, weighed and snap frozen separately indry ice (at −70° C. plus or minus 10° C.). Tissues samples are stored at−70° C. (plus or minus 10° C.) for an optional HPLC analysis of dopamineand its metabolites. If this option is not taken, the striata will bedestroyed.

Coronal serial sections of the entire mesencephalon were cut on acryostat at 50 μm intervals. Sections were collected free-floating inwell-plates containing cryoprotectant solution, which were then storedat −20° C. until the day of TH immunohistochemical processing.

TH immunohistochemistry was performed as follows on every fourthsection. Tissue sections were taken from the −20° C. freezer, left toadjust to room temperature, and then rinsed in PBS solution. Endogenousperoxidase was inhibited by incubating in PBS containing 0.3% H₂O₂ for10 minutes. Following this, sections were washed in PBS, incubated inPBS 4% normal horse serum (NHS) and 0.3% Triton X-100 for 30 minutes,for the blockade of non-specific antigenic sites. Sections were thenincubated overnight at room temperature in antibody dilutant+primaryantibody for tyrosine hydroxylase (TH) (anti-TH affinity isolatedantibody, Sigma T8700) at a dilution of 1/10,000. Sections were thenrinsed thoroughly in PBS and incubated for 30 minutes in ImmPRESS Igperoxidase polymer detection reagent (Vector MP7401). Following this,sections were thoroughly washed with PBS Immunological staining was thenrevealed with 3,3′-Diaminobenzidine (DAB)/Tris/H₂O₂ kit (Vector SK4100).After one minute, revelation was stopped with several PBS washes.Sections were mounted and counterstained with 0.1% cresyl violet.

Unbiased stereological analysis was used to estimate the number ofTH-immunopositive (TH+) neurons (Mercator, Explora Nova, La Rochelle,France). The boundaries of the SN were determined by examining the sizeand shape of the different TH+ neuronal groups. The volume wascalculated by using the formula: V=ΣS td; where ΣS is the sum of surfaceareas, t is the average section thickness and d is the number of slicesbetween two consecutive sections measured. One in every 4 sections wasused; optical dissectors were distributed using a systematic samplingscheme. Dissectors (50 μm length, 40 μm width) were separated from eachother by 150 μm (x) and 120 μm (y). The following formula was used toestimate the number of TH+ neurons: N=V(SN) (ΣQ−/ΣV(dis)); where N isthe estimation of cell number, V is the volume of the SN, ΣQ− is thenumber of cells counted in the dissectors, and ΣV(dis) is the totalvolume of all the dissectors. Mean estimated number of neurons and SEMwas then calculated for each group

All statistical analyses were performed using Graphpad prism version 7.All data are presented as mean±the standard error of the mean (SEM). Theeffect of AOL was analysed with a one-way ANOVA followed by Dunnett'smultiple comparisons post-hoc analysis. A P value of less than 0.05 wasconsidered significant.

Results

There was a significant effect of treatment on the number of TH+ cellsin the SN (F2,29=10.94, p<0.001, FIG. 10). The number of TH+ cells inthe SN was reduced by 39% (p<0.001) in MPTP-treated compared tovehicle-treated animals. Following administration of AOL, the number ofTH+ cells in the SN was increased by 44% (p<0.01) compared to vehicle inthe MPTP-treated mice.

Conclusion

AOL treatment for 11 consecutive days, at a dose of 5 mg/kg, has asignificant neuroprotective effect compared to vehicle, in preventingthe MPTP-induced reduction in TH+ cells in the SN, resulting in 44% morecells surviving in the SN with the administration of AOL.

These data suggest that AOL treatment can protect the dopaminergicneurons in the substantia nigra from MPTP intoxication.

Example 5: Effect of AOL in Cardiovascular Disease: Ischemia-ReperfusionInjury

The present study aims at evaluating the capacity for AOL to protectperfused rat heart from the damages occurring after global ischaemia andreperfusion.

The consequences of 30 minutes' global ischaemia followed by 120minutes' reperfusion (FIG. 11) on contractility and tissue viabilitywere studied on isolated perfused rat heart pretreated or not (controlvehicle) with 10 μM AOL.

Material and Methods

All procedures conformed to the UK Animals (Scientific Procedures) Act1986 and the Guide for the Care and Use of Laboratory Animals publishedby the National Institutes of Health (NIH Publication No. 85-23. revised1996). Male Wistar rats (250-300 g) were anesthetized by 3% isoflurane,heparinized and euthanized by a lethal IP injection of pentobarbital(130 mg/kg). Hearts (˜0.95 g of fresh weight) were rapidly harvested andplaced into ice cold Krebs-Henseleit buffer containing (in mmol/L): NaCl118, NaHCO₃ 25, KCl 4.8, KH₂PO₄ 1.2, MgSO₄ 1.2, glucose 11 and CaCl₂1.8; gassed with 95% O₂/5% CO₂ at 37° C. (pH 7.4). Langendorff heartperfusions were performed (Garlid, K. D. et al., 2006. Am. J. Physiol.Heart Circ. Physiol. 291(1):H152-60) and contractility was assessed bycontinuous measurement of the rate pressure product (RPP) thanks to aballoon placed in the left ventricle and connected to a pressuretransducer. Hearts were perfused in a constant flow mode (12 mL/min).After 10 minutes for stabilization followed by 10 minutes of treatmentwith the vehicle (Control) or 10 μM AOL solution, global normothermicischaemia was induced by halting perfusion flow for 30 minutes whileimmersing the heart in perfusion buffer at 37° C. At the end of thereperfusion period, hearts were stained to assess infarct size, orfreeze-clamped using liquid-nitrogen cooled tongues. In the latter case,hearts were grinded under liquid nitrogen, and stored at −80° C. forfurther analyses.

At the end of the reperfusion period, hearts were stained withtriphenyltetrazolium chloride (TTC): hearts were perfused for 7 minutesat 13 mL/min with a 12% (w/v) TTC solution in order to get a 1% finalconcentration in the heart. Hearts were then detached from the cannulaand incubated for an additional 4 minutes at 37° C. before being slicedperpendicular to the longitudinal axis into 6 slices. The slices werethen treated in 4% (w/v) formalin solution overnight at 4° C. andweighed, before both sides of each slice were photographed. The surfaceof the necrotic and at risk areas of each side were determined on eachphotography by planimetry (AlphaEase v5.5), and infarct size wasexpressed as a percentage of the total cross-sectional area of theheart, since total heart was subjected to ischemia.

Data from 6 independent preparations are expressed as means±SEM. The nnumber in each group being smaller than 20, the distribution wasconsidered as non-normal and consequently a non-parametric Mann-Whitneytest (SPSS statistics 17.0) was performed to compare the two groups.Results were considered statistically significant if the p-value wasbelow 0.05.

Results

FIG. 11 presents the evolution of the RPP—considered here as a surrogateof heart contractility—during the critical phase of the reperfusionfollowing ischaemia. Clearly, AOL improves the contractility and afteran identical evolution as compared to the control hearts treated withAOL, showed an improvement of contratility which was about three timeshigher than control hearts after 2 hours of reperfusion. At this time,hearts were prepared for TTC staining to assess tissue viability. Thehigher contractile activity for AOL hearts was confirmed by TTCstaining, and photographs of the slices of treated and non-treatedhearts (data not shown) clearly show that AOL induced an importantprotection of cardiac tissue. This protection has been analyzed morethoroughly and the results are presented in FIG. 12. The infarctsize—damaged tissue—is expressed as percentage of the total surface foreach independent experiment together with the mean value for AOL-treatedand non-treated hearts. Results clearly show that AOL highlysignificantly protects cardiac tissue from ischaemia/reperfusiondamages. In fact, about 50% of infarcted tissue was rescued bypre-treatment with AOL (FIG. 12).

Conclusion

These results extend, under ex vivo (living organ) conditions, the roleof AOL as an inhibitor of mitochondrial ROS production, most probably atthe level of complex I.

They also evidence the therapeutic interest of AOL for tissue protectionagainst ischaemia/reperfusion damages, not only in heart but also in anytissue subjected to ischaemia.

Example 6: Effect of AOL in a Cardiovascular Disease: Cardiac Toxicityof Anthracyclines

The present study aims at evaluating the effect of AOL in a model ofcardiac toxicity of anthracyclines. This was assessed by administeringanthracycline-derived anti-cancer drugs, together with AOL, to 10 weekold rats for 14 to 17 days.

Material and Methods

The studies were performed on Sprague-Dawley rats aged 10 weeks anddifferent treatments were administered intraperitoneally for 14 to 17days, before collection of the heart for analysis. To respect the “threeRs principle” in animal experimentation, the number of group tested waslimited as much as possible, in particular by focusing the experimentson one anthracycline molecule only, namely Doxorubicine, and bycomparing the effect of AOL to one alternative protective molecule only,namely Dexrazoxane.

At the end of the experiment, the heart of the rats treated will beremoved and cardiac function will be studied exhaustively afterperfusion of these hearts in a Langendhorf system to determine thefunction cardiac affected by doxorubicin and whether AOL treatment isefficient.

The study comprises 5 different groups for 8 rats each:

-   -   1—Control group. Rats received the vehicle only, composed of 5%        DMSO+95% NaCl 0.9%, twice a day (morning and evening) for 17        days;    -   2—Doxo group. Rats received Doxorubicine at a dose of 3 mg/kg        (ip), every two days (morning), from day 3, for 14 days. Rats        received vehicle only for every other injections;    -   3—Dexra group. Rats were treated with Dexrazoxane (reference        protecting agent) at a dose of 30 mg/kg ip simultaneously with        Doxorubicin at a dose of 3 mg/kg ip (according to the dosage        ratio recommended by the French Regional Health Agency “ARS” in        2011), every two days, from day 3, for 14 days. Rats received        vehicle only for every other injections;    -   4—AOL group. Rats were treated with AOL and Doxorubicin:        -   4 mg/kg ip of AOL, mornings and evenings, for 72 hours            preceding the first injection of Doxorubicin;        -   on the days of Doxorubicin injection (based on the Doxo            group): 4 mg/kg ip AOL together with the Doxorubicin            injection, followed 90 minutes later by a second injection            of AOL at a dose of 4 mg/kg ip;        -   on the days without Doxorubicin injection: 4 mg/kg ip of            AOL, mornings and evenings.    -   5—AOL/Carv/Enal group. Rats were treated similarly than rats        from the AOL group here above. AOL injections were supplemented        with a classical treatment for cardiac insufficiency (Carvediol,        a β-blocker, at a dose of 1 mg/kg, and Enalapril, a vasodilator,        at a dose of 0.5 mg/kg).

Example 7: Effect of AOL in in a Cardiovascular Disease: PulmonaryHypertension

The present study aims at studying the role of mitochondria in thepulmonary vasculature physiology and providing a new alternativetreatment of pulmonary hypertension. This disease is characterized byincreased pulmonary arterial pressure and remodeling of pulmonaryarteries (PA), leading to increased pulmonary vascular resistance,hypertrophy of the right ventricle, right heart failure and ultimately,death.

Pulmonary hypertension can be divided into five groups, among which thegroup 1 corresponds to pulmonary arterial hypertension. As for group 3,it includes pulmonary hypertension due to lung diseases (such as chronicobstructive pulmonary disorder) and/or alveolar hypoxemia.

To address the issue of the effect of AOL, two different rat models wereused: a hypoxia model and a monocrotaline-induced model, that sharepathophysiological characteristics with group 3 and group 1 pulmonaryhypertension, respectively.

Material and Methods

Male Wistar rats (300-400 g) were separated into 3 groups and used 4weeks later:

-   -   the first group (control or normoxic rats—N rats) was housed in        ambient room air;    -   the second group (chronic hypoxic rats—CH rats) was exposed to        chronic hypoxia for 3 weeks in a hypobaric chamber (50 kPa), and    -   the third group (MCT rats) was injected with a single        intraperitoneal dose of monocrotaline at a dose of 60 mg/kg. MCT        (Sigma, St Quentin Fallavier, France) was dissolved in an equal        volume of HCl (1 M) and NaOH (1 M).

In each group, some animals were treated with AOL (Sulfarlem, EG LaboEurogenerics. Crushed tablets mixed with food, fed ad libitum) and someother animals were untreated. Eaten food was weighed every day toestimate the AOL dose administered. 10 mg/kg/day was thus administeredduring the 3 weeks of experiment for the second and third groups.

For each condition, 7 to 10 rats were used. All animal care andexperimental procedures complied with the recommendations of theFederation of European Laboratory Animals Science Association, and wereapproved by the local ethics committee (Comite d'éthique régionald'Aquitaine—referenced 50110016-A).

Pulmonary hypertension was assessed by measuring both the mean pulmonaryarterial pressure (mPAP) and right ventricle hypertrophy. To measurePAP, N, CH and MCT rats were anesthetized with pentobarbital sodium(Centravet) by intraperitoneal injection (60 mg/kg) and mPAP wasmeasured, in closed-chest rats, through a catheter inserted in the rightjugular vein, then through the right atria and the right ventricle intothe pulmonary artery, and attached to a Baxter Uniflow gauge pressuretransducer.

Right ventricle hypertrophy was estimated by the ratio of rightventricle (RV) to left ventricle plus septum (LV+S) weight (Fultonindex).

Pulmonary arteries (PA) remodeling was assessed by measuring thepercentage of the PA medial thickness from sections of paraffin-embeddedlung. Lung sections were first stained with hematoxylin and eosin (VWR)according to common histological procedure. On each section, threegroups of 10 intracinar arteries with different cross-sectionaldiameters were observed to evaluate medial wall thickness (namelycross-sectional diameters under 50 μm, between 50 to 100 μm and between100 to 150 μm).

Results

Results are expressed as mean±SEM of n independent observations. Alldata were analyzed using a non-parametric test for unpaired samples(Mann-Whitney test). FIG. 13 shows the effect of AOL on pulmonaryarterial pressure (FIG. 13A) and heart remodeling (FIG. 13B). nindicates the number of rats for mPAP and Fulton index measurements. Allbar graphs and statistics were performed with Graphpad PRISM software(v6, Graphpad Software). P<0.05 were considered significant. As seen,AOL had no significant effect on the control group (N rats). However,mean pulmonary arterial pressure was decreased in MCT rats treated withAOL, and even more significantly in CH rats treated with AOL. AOLtreatment had however no effect on the Fulton index.

FIG. 14 shows the effect of AOL on pulmonary arteries remodeling. nindicates the number of vessels analyzed for % of medial thicknessmeasurement. All bar graphs and statistics were performed with GraphpadPRISM software (v6, Graphpad Software). P<0.05 were consideredsignificant. AOL shows a significant effect in CH rats, in whichpulmonary arteries diameter was reduced by 30%.

Conclusion

AOL treatment, at an oral dose of 10 mg/kg/day, has a significant effectin the prevention and/or treatment of pulmonary hypertension in vivo, inparticular in group 3 pulmonary hypertension. Results indeed show asignificant improvement of clinical symptomatology.

These data suggest that mitochondria play a major role in the pulmonaryvasculature physiology, and extend the use of AOL to the treatment ofpulmonary hypertension.

Example 8: Effect of AOL in Aging Disease and Progeroid Syndromes:Macular Degeneration

The present study aims at evaluating the capacity for AOL to protectretina against progressive degeneration.

Material and Methods

Rats bred under cyclic low-intensity lighting were transferred to cyclichigh-intensity lighting for one week and divided into 3 groups(non-treated animals, vehicle-treated animals and AOL-treated animals).Treated animals received injections of vehicle or AOL at a dose of 6mg/kg/day, three times a day for the 7 days of the transfer (30 minutesbefore light-ON; at 01.00 μm; at 09.00 μm). After one week, animals weretransferred in the dark (D0).

A control group (“untransferred”) was not transferred to cyclichigh-intensity lighting but received the same treatment as describedabove: injections of vehicle or AOL three times a day for 7 days,followed by a transfer in the dark (D0).

On the day following the transfer in the dark (D1), a firstelectroretinography is performed. It measures the electrophysiologicalsignal which is generated by the retina, in response to a lightstimulation. It is typically characterized by two waves, namely a-waveand b-wave. a-wave represents the initial corneal-negative deflection,derived from the cones and rods of the outer photoreceptor layers. Itreflects the hyperpolarization of the photoreceptors due to closure ofsodium ion channels in the outer-segment membrane. b-wave represents thecorneal-positive deflection, derived from the inner retina(predominantly Muller and ON-bipolar cells). Analysis of theelectroretinogram consists in measuring the amplitude and/or latency ofthese waves, as a function of the intensity of the light stimulation.a-wave amplitude, for a given light stimulation intensity, depends onthe number of photoreceptors; whereas amplitude of b-wave, for a givenlight stimulation intensity and a given number of photoreceptors,indicates the signal transmission efficiency.

After D1's electroretinography, animals were transferred back undercyclic low-intensity lighting conditions, and a secondelectroretinography is performed at D15.

Animals were then sacrificed for histological analysis. The thickness ofthe various layers of the retina, in particular the thickness of theouter nuclear layer (ONL) and inner nuclear layer (INL) were measured(in μm, from the optical nerve and every 0.39 mm in the superior andinferior poles of the optic disc).

Results

Histological analysis is reported on FIG. 15. It shows that, in thecontrol group (“untransferred”), treatment with AOL has no effect onONL's thickness (FIG. 15A). This suggests that AOL does not have a toxiceffect on retina's photoreceptors.

On the contrary, transfer in cyclic high-intensity lighting conditions(“transferred”) induces a significant decrease (by half in some areas)of the ONL, in non-treated animals. AOL however tends to protect the ONLagainst light-induced damages. Histological analysis has indeed shown asignificant increase of the thickness of the ONL in AOL-treated/cyclichigh-intensity lighting-exposed animals (FIG. 15B).

Conclusion

AOL treatment has a significant protective effect against light-induceddamages on the retina. In particular, the thickness of the retina wasshown to be preserved as compared to non-treated animals, afterprolonged cyclic high-intensity lighting exposure.

Example 9: Effect of AOL in Diseases Related to MitochondrialDysfunctions

The present study aims at testing the effect of AOL in vivo, in a modelof oxidative phosphorylation dysfunction.

Material and Methods

Mice deficient in mitochondrial Mn-Superoxide Dismutase (Sod2-KO) on aCD1-background were used. This genetic alteration leads to an adversephenotype and the death of animals at an average of 8 days old.Mitochondrial superoxide dismutase is a free radical scavenging enzymewhich transforms superoxide (highly reactive) into hydrogen peroxide(less reactive), that could then cross mitochondrial membranes and bedetoxified by matrix and cytosolic anti-oxidant systems. The aim of thisstudy was to test if AOL could rescue the Sod2-KO phenotype through itsactivity on I_(Q) superoxide production.

After birth, pups were genotyped (3 day-old) and the litter size wasreduced to 6 pups per cage. Animals were then treated (AOL inKolliphor®-5 mg/kg) or not (Kolliphor® only, noted KOL below). Thechoice of the dosage was mainly driven by the solubility limit of thecompound (2.8 mM in Kolliphor®) and the maximum injectable volume inpups (6 to 7 μL per gram body mass). Two studies were conducted on twodifferent generations from the same parents: lifespan; and succinatedehydrogenase activity in heart (SDH) and Oil Red O staining in liver.Animals were weighed and injected (intra-peritoneal) daily.

Succinate dehydrogenase activity is a marker of superoxide in themitochondrial matrix. Thus, a lack of SOD2 is associated with a decreaseof SDH activity in heart. The aim of this experiment was to test whetheror not AOL could restore SDH activity in KO mice.

Oil Red O staining is a marker of lipid that has been shown toaccumulate in Sod2-KO liver. However, the direct link betweensuperoxide/hydrogen peroxide production and liver lipid accumulation isnot established. The aim of the study was to test the potency of AOL toprevent liver lipid accumulation in Sod2-KO mice.

Results

Lifespan

4 groups were constituted:

-   -   1—WT-KOL (n=7), a group of wild-type mice treated with the        vehicle only,    -   2—WT-AOL (n=17), a group of wild-type mice treated with AOL,    -   3—KO-KOL (n=2), a group of Sod2-KO mice treated with the vehicle        only,    -   4—KO-AOL (n=4), a group of Sod2-KO mice treated with AOL.

Animals were injected once a day from 3 days old until their death.

FIG. 16A and FIG. 16B show the evolution of body weight (A) andpercentage of initial body weight. These results show that body weightand body weight gain were lower in KO-mice than in WT-mice. Treatmentwith AOL however tends to alleviate this effect as seen from day 8 to12, suggesting a potential beneficial effect of the compound.

FIG. 16C shows the survival proportion of Sod2-KO mice whether they weretreated with AOL or not. As expected in view of the above results, bothmedian lifespan and maximal lifespan were slightly improved by AOLtreatment in KO mice, with AOL-treated mice living up to 2 days longeras compared to untreated mice, supporting a beneficial effect of AOL.

SDH Activity in Heart & Oil Red O Staining

5 groups were constituted:

-   -   1—WT-non-injected (n=6), a group of untreated wild-type mice;    -   2—WT-KOL (n=6), a group of wild-type mice treated with the        vehicle only;    -   3—WT-AOL (n=6), a group of wild-type mice treated with AOL;    -   4—KO-KOL (n=4), a group of Sod2-KO mice treated with the vehicle        only;    -   5—KO-AOL (n=6), a group of Sod2-KO mice treated with AOL.

In this study, animals were treated daily (5 mg/kg) from day 3 to day 5.Heart and liver were harvested at day 6.

As expected, SDH activity tended to decrease (not significant) in KOcompared to WT animals. However, AOL showed only a very slight increasein SDH activity in KO mice, but could not restore SDH activity to thelevels of WT mice (FIG. 17).

FIG. 18 shows lipid droplets average size (Panel A), density (Panel B)and area (Panel C). Untreated KO mice exhibited a high lipid contentphenotype compared to WT animals. In AOL-treated KO mice however, lipiddroplets density decreased as compared to untreated animals. Moreimportantly, these results also show that AOL treatment was able torestore the total lipid area in KO mice, consistent with on-targetsuppression of mitochondrial superoxide production in vivo in Sod2-KOmice.

Conclusion

In vivo studies show encouraging results. Although AOL treatment couldnot fully counteract the effects of SOD2 depletion in mice, results showthat lifespan could still be extended by a couple of day as compared tountreated KO animals, together with an alleviation of the decrease inbody weight gain. This suggests a potential effect of AOL.

AOL bioavailability is known to be very short. Thus, treatment withhigher doses might lead to improve AOL effects in these experiments.However, constitutive KO remains a high adverse phenotype to rescue withonly one very specific treatment and may require synergic action withother drugs.

In vivo, results also showed that AOL could restore lipid content and/orprevent lipid accumulation in liver of Sod2-KO mice.

Example 10: Effect of AOL in an Autoimmune Disease: Scleroderma

The present study aims at testing the effect of AOL on fibroblasts frompatients with scleroderma. Scleroderma is a chronic systemic autoimmunedisease characterized by an increased synthesis of collagen, damages tosmall blood vessels, activation of T lymphocytes and production ofaltered connective tissues.

Material and Methods

Fibroblasts from both a healthy donor and a patient with scleroderma arecultured in flasks, in complete DMEM medium (10% FCS, 1% antibiotic).After 6-hour adhesion, cells are deprived of serum overnight.

AOL is extemporaneously prepared. AOL was weighed and dissolved in DMSOat 5 mg/mL. This stock solution was further diluted to 10 and 5 mMfinal, in DMSO. AOL was further diluted in complete DMEM medium, toreach final concentrations of 40, 20 and 10 μM.

Cultured cells were contacted with AOL at 40, 20 and 10 μM. Controlcells were contacted with complete DMEM medium, supplemented with DMSO(0.2%) and N-acetylcysteine (3 mM). Cells are incubated under normoxicconditions (37° C., 20% 02) and under hypoxia (37° C., 1% 02) for 6 or24 hours.

For MMP-1, MIP and MCP secretion analysis, the culture supernatant isharvested, aliquoted and stored at −20° C. for dosage. MMP-1 isquantified by ELISA (Abcam), according to the manufacturer'sinstructions. MCP-1 and MIP-1α concentrations are quantified by CBA(Cytometric Bead Array, Biolegend).

For MMP1, collagen and CCl2 expression analysis, cells are detached withtrypsin and washed with PBS. The cell pellet is then resuspended inlysis buffer, and RNA extraction is carried out according to themanufacturer's instructions (Nucleospin RNA Plus, Macherey Nagel). 1 μgof RNA is retro-transcribed (GoScript, Promega), then diluted 10-foldbefore SYBR Green qPCR (SYBR qPCR Premix Ex Taq, Takara) in a BioRadCFX384 PCR machine. Primers for MMP-1, Col1A2 and CC12 are used tomeasure genes of interest; primers for Ppia, RPLP0 and EEF1A1 are usedto measure reference genes.

The invention claimed is:
 1. A method for treating or preventing freeoxygen radicals-related liver diseases in a subject in need thereof,said method comprising administering to said subject an inhibitor ofproduction of reactive oxygen species (ROS), wherein said inhibitor ofproduction of ROS is anethole trithione, and wherein said free oxygenradicals-related liver disease is selected from the group consisting ofdiabetes, alcoholic fatty liver disease, non-alcoholic fatty liverdisease, liver inflammation in hepatitis C and cirrhosis.
 2. The methodaccording to claim 1, wherein said free oxygen radicals-related liverdisease is diabetes.
 3. A method for increasing insulin secretion in asubject in need thereof, said method comprising administering to saidsubject an inhibitor of production of reactive oxygen species (ROS),wherein said inhibitor of production of ROS is anethole trithione. 4.The method according to claim 3, for increasing glucose-stimulatedinsulin secretion (GSIS).
 5. The method according to claim 3, whereinthe subject in need thereof is affected with an insulin secretiondeficiency.
 6. The method according to claim 3, wherein the subject inneed thereof is affected with a free oxygen radicals-related liverdisease selected from the group consisting of diabetes, alcoholic fattyliver disease, non-alcoholic fatty liver disease, liver inflammation inhepatitis C and cirrhosis.
 7. The method according to claim 3, whereinthe subject in need thereof is affected with diabetes.
 8. A method fordecreasing body weight and/or food intake in a subject in need thereof,said method comprising administering to said subject an inhibitor ofproduction of reactive oxygen species (ROS), wherein said inhibitor ofproduction of ROS is anethole trithione, and wherein decreasing bodyweight comprises decreasing fat mass and/or blood glucose levels in thesubject in need thereof.
 9. The method according to claim 8, wherein thesubject in need thereof is affected with a free oxygen radicals-relatedliver disease selected from the group consisting of diabetes, alcoholicfatty liver disease, non-alcoholic fatty liver disease, liverinflammation in hepatitis C and cirrhosis.
 10. The method according toclaim 8, wherein the subject in need thereof is affected with dietaryobesity.